What is QIAzol

Real-time analysis of the transcription factor binding, transcription, translation and sales, to display global events during cellular activation

Summary

This protocol describes the combinatorial use of ChIP-Seq, 4sU-Seq, total RNA-Seq and ribosome profiling for cell lines and primary cells. Tracking of changes in the transcription factor is mandatory, de Novo Enables transcription, RNA processing, turnover and translation over time and shows the full course of events in activated and / or rapidly changing cells.

Abstract

After activation, cells rapidly change their functional programs and thus their gene expression profile. Massive changes in gene expression occur, for example, in cell differentiation, morphogenesis, and functional stimulation (e.g. activation of immune cells), or after exposure to drugs and other factors from the local environment. Depending on the stimulus and cell, these changes occur quickly and at every level of gene regulation. Viewing all the molecular processes a cell reacts to a certain type of stimulus / drug is one of the most difficult tasks in the field of molecular biology. Here we describe a protocol that enables the simultaneous analysis of multiple layers of gene regulation. We compare, in particular, binding transcription factor (chromatin immunoprecipitation sequencing (ChIP-Seq)), de-novo -Transcription (4-thiouridine sequencing (4sU-Seq)), mRNA processing, as well as turnover and translation (ribosome profiling). By combining these methods, it is possible to display a detailed and genome-wide procedure.

Newly transcribed RNA sequencing is particularly recommended when analyzing fast adapting or changing systems, as this shows the transcriptional activity of all genes during the time of 4sU exposure (regardless of whether it is up or down regulated). The combinatorial use of total RNA-Seq and ribosome profiling also enables the calculation of the RNA turnover and translation rates. Bioinformatic analysis of high throughput sequencing results allows many means for analysis and interpretation of the data. The data generated can also track, co-transcriptional and alternative splicing, to name a few.

The combined approach described here can be used for different model organisms or cell types, including primary cells. We provide detailed logs for each method used, including quality controls, and discuss possible problems and pitfalls.

Introduction

In recent years, RNA sequencing (RNA-Seq) has become the standard tool of all expressed RNAs within a cell or organism1analyze. However, in order to understand the entire process of cells adapting in response to a particular stimulus / drug, it is necessary to fully establish all the underlying processes, editing, turnover and translation of mRNA transcription up. Short-term changes in RNA transcription can hardly be measured from total RNAseq, since changes in total RNA depend on factors such as. RNA half-lives and transcriptional activity are a poor template to reflect the adaptation of cells to environmental effects2,3. In fact a variety of new sequencing techniques have been developed that allow an analysis of the various steps in the process of gene regulation4 Allow combination in the right way. This protocol describes how some fairly easily applicable sequencing techniques combine to allow tracking of the regulation of the essential layers of the mRNA in a comparative manner. For the analysis of transcriptional activity, a variety of methods have been described, such as GAP analysis of gene expression (CAGE)5, native stretching transcript sequencing (NET-Seq)6and genome-wide nuclear wake (GRO-Seq)7,8, as well as bromouridine sequencing (Bru-Seq) and 4-thiouridine sequencing (4sU-FF), use the metabolites that are incorporated into newly transcribed RNA9,10to name a few. While CAGE identifies the exact transcription start site, NET-Seq and GRO-Seq provide more precise information about reading directions and 4sU-Seq (this is the method described here) only recognizes newly transcribed RNA. However, 4sU-Seq is very sensitive and can be applied in different time frames to measure quantitative transcriptional activity, actively changing cells as well as quantitative changes in mRNA processing (which occurs within minutes)9, 11,12. In addition, 4sU-Seq is ideally combined with RNA-Seq RNA turnover rates for genes9to calculate. The mRNA is transcribed by RNA polymerase II (RNAPII), which in turn is influenced by a number of factors, such as: B. transcription factors, histone modifications and general activators / repressors, which can even include the transcriptional complexes. To test how many gene / organizer / enhancer regions are bound by a factor, ChIP-Seq has developed, which is now the standard method for this purpose, due to the many antibodies available on the market13. However, although ChIPseq gives clear information on where bind regulate factors, it does not reflect when there are indeed changes in transcription14 leads. Carrying out ChIP-Seq with 4sU-Seq is therefore the ideal combination for such biological questions. Regulation of gene expression can also occur at a later date, as mRNA and protein levels are not necessarily15,16 correlatedThat may indicate significant regulation on the translational or post-translational level, depending on the context. In 2011 ribosome profiling had initially been combined with RNA-Seq and is now the method of choice to quantify changes that occur rapidly in protein, since there are still some sensitivity limits with mass spectrometry17. In fact, conversion rates obtained from such methods have been shown to provide a relatively good estimate of changes in protein level (at least measured for long-term changes) and allow an even more detailed view of the translation process, z. B. the determination of the start page and alternative translation framework17. The combinatorial use of all four methods can be used in steady state, between different cell types, or in the time series of a rapidly changing cell11to experiment. This insert provides a genome-wide overview of changes in transcription factor binding affecting RNA transcription, editing, and translation.

Protocol

All methods are in accordance with and adhering to the Helmholtz Zentrum München institutional, federal and state guidelines.

1. Preparation

  1. Make a detailed plan of the experimental setup including a schedule of when to add 4sU to the cell culture medium and when to harvest the cells for each method. Depending on the biological question, carefully weigh up the points in time for example drawing, 4sU marking time and concentration (Table 1).
    NOTE: Check the effects of 4sU on cell viability and emphasize the answer in advance (see "Verify 4sU Labeling Optimal Conditions" representative and illustration 1). It is recommended to carry out a preliminary test of the individual methods with at least one sample. Check that the quality and amount of RNA / DNA is sufficient for deep sequencing (see dedicated parts of the protocol), and quick but gentle treatment of the cells during the experiment.
  2. Calculate the number of cells required by each method for each point in time (see Table 2 for a rough estimate of primary T cells). Also consider sequencing fewer samples of total RNA than just the 4sU RNA (z. B.only at the time of translation prices or RNA sales prices should be calculated). To confirm and verify the significance of the results (recommended), create at least one biological replicate.
    Note: It is important that samples from the same starting point pool of cells must be used for all methods and successive time points. At least one dedicated researcher for each method is recommended.
  3. Prepare everything necessary in advance (e.g., regularly 4sU cycloheximide, mammalian lysis buffer and 1% formaldehyde). After adding 4sU, avoid exposing the cells to bright light, as this will lead to cross-linking of the 4sU labeled RNA to cellular proteins18can lead.
  4. Bundle all cells of interest in a flask just before treatment (Figure 2). Counting the cells (e.g.with a hemocytometer) and use the required amount for the untreated control of the individual methods (do not forget to also mark the untreated control for 4sU-Seq with 4sU). Treat remaining cells and immediately divide the required number of cells for each time point and method. Treat cells as soon as possible for stress due to changes in temperature or CO2 -Concentration to minimize.
    Note: for example, samples are taken 1 h, 2 h, 3 h after the treatment, then a quarter of the cells are used for the untreated control and three quarters are used for the treated control.

2. 4sU marking

Note: This protocol has been changed by Rädle, Et al. 19 refer to their protocol for more information on metabolic labeling with 4sU. For all methods and consecutive time points, samples must come from the same starting point pool of cells.

  1. Start of marking
    1. Thaw regularly for 4 seconds just before use. At each time point, add 4sU directly to cells of interest-containing medium (see Table 2 for recommended T cell counts, at least 60 µg RNA per time point), mix, and place back in the incubator. Discard any remaining 4sU (do not refreeze).
    2. At the end of the label, collect cells (z.B. Cell scraper) and centrifuge at 330 x g for 5 min at 4 ° C in polypropylene tubes (to withstand the high g-forces). Aspirate medium and add reagent for RNA isolation (≥1 mL per 3 x 106 Cells, see Materials for recommendation which to use) around each tube. Fully resuspend pellet (≥1 mL per 3 x 106 Cells), incubate for 5 min at room temperature (RT) and freeze the samples at -20 ° C. Samples can be stored at -20 ° C for at least 1 month.
      Attention: The reagents for RNA isolation are extremely dangerous if always in contact with skin or eyes. Treat them with care and observe the safety instructions.
  2. RNA preparation with modified RNA isolation protocol
    1. 0.2 mL chloroform per 1 mL reagent for RNA isolation, and mix thoroughly by shaking for 15 S. Continue as described in the metabolic labeling protocol (steps 1-12, 2nd RNA preparation using the modified Trizol protocol) by Rädle Et al. 19
    2. Measure RNA concentration (see Table of materials), according to the manufacturer's instructions. Use this RNA for total RNA-Seq (see step 3. Total RNA-Seq) or store at -80 ° C for at least 1 month.
  3. Thiol-specific biotinylations of newly transcribed RNA
    1. Start with 30-80 µg of total cellular RNA. 60 µg of RNA should result in sufficient amounts of newly transcribed RNA.
    2. Prepare labeling reaction. Pipette in the following order (per µg RNA): 1 µL 10 x biotinylation buffer, 7 µL RNA (diluted with 1 µg RNA in nuclease-free H2O), and 2 µL biotin-HPDP (1 mg / mL). Biotin-HPDP last and mix immediately by pipetting. Wrap pipes with aluminum foil to avoid bright light. See the discussion for an alternative to biotin-HPDP. Incubate 1.5 h with rotation at RT.
    3. Centrifuge corresponding 2 mL tubes (see Table of materials) at 15,000 x g for 2 min.Pipette all of the biotinylated RNA in the pre-spun 2 mL tube, add an equal volume of chloroform and mix vigorously. Incubate for 2-3 min until the phases start to separate and air bubbles start to disappear.
    4. Centrifuge at 15,000 x g for 15 min at 4 ° C. Carefully transfer the upper aqueous phase into a new tube.
    5. Repeat steps 2.3.3. and 2.3.4. once. Add 10% of the volume of NaCl (5 M) and an equal volume of isopropanol in the aqueous phase. Centrifuge at 20,000 x g for 20 min at 4 ° C. Discard the supernatant.
    6. Add an equal volume of 75% freshly made ethanol. Centrifuge at 20,000 x g. Discard the supernatant, turn briefly and remove the remaining ethanol. 30-100 µL of H.2O (use 1 µL H2O per 1 µg input RNA from step 2.3.1) whirl up fully by mixing the pipette.
    7. Check the integrity of the RNA by electrophoretic analysis or take an aliquot and confirm it later.
  4. Separation of newly transcribed (labeled) and existing (unlabelled) RNA
    1. Remove paramagnetic beads (see Table of materials) from 4 ° C memory and let it stand for at least 30 minutes to bring it to room temperature. Heat 4sU wash buffer (3 mL per sample) to 65 ° C.
    2. Prepare 100 mM dithiothreitol (DTT) solution. Weigh DVB-t on ultra-fine scales and add the required amount of nuclease-free water. Always prepare fresh. Use 200 µL per sample.
    3. Warm biotinylated RNA samples (1 µg / µL) to 65 ° C for 10 min to denature and immediately place on ice. Add biotinylated RNA 100 µL streptavidin beads and incubate for 15 min with rotation at RT.
    4. To place a corresponding column (see Table of materials for recommendations) place each sample in the magnetic stand and pre-equilibrate each column with 1 mL of room temperature 4sU wash buffer.
      Note: this takes about 5-10 minutes. If any of the crevices do not start emptying after 5 minutes, gently press the support with a gloved finger.
    5. Apply an RNA / bead mixture to the center of each column. Discard flow-through unless unlabeled RNA needs to be restored. If so, collect the perfusion and at least the first wash. Perform the restoration of RNA like wheels Et al. described (Step 1-7, 7. Restoration of the unlabeled, unbound RNA)19.
    6. Wash three times with 0.9 mL of 4sU wash buffer (prewarmed to 65 ° C from step 2.4.2.) And 0.9 mL of RT 4sU wash buffer, respectively.
    7. Use paramagnetic beads to recover newly transcribed RNA. Pipette 400 µL of well-dispersed RT paramagnetic beads in one tube per sample and place under each column. Elute newly transcribed RNA with 100 µL 100 mM DTT. Wait 3 minutes and perform a second elution with 100 µL 100 mM DTT. (Optional: Carry out elution and restoration as described by Rädle Et al.) 19
    8. Mix re-transcribed RNA / beads thoroughly with pipette for 10 mixing times and follow the manufacturer's guideline. Elute RNA in 11 µL nuclease-free H2O. Quantify RNA with a suitable fluorometer (see Table of materials). RNA can be stored at -80 ° C for at least 1 month.
      Note: Newly transcribed RNA can be used to prepare the cDNA libraries for next generation sequencing (see Table of materials for a suggestion of which kit to use) or further downstream analysis. 100-500 ng RNA is sufficient for most library preparation kits (see discussion).

3. total RNA-Seq

  1. Take RNA directly from 4sU labeled RNA after RNA preparation with the modified reagent for RNA isolation protocol for total RNA-Seq (see point 2.2.2.)
  2. Prepare the library by diluting one RNA aliquot to a final concentration of 50-100 ng / µL. use the same library kit to prepare the newly transcribed RNA. 100-500 ng of RNA is sufficient for most library preparation kits.

(4) Ribosome profiling

Note: For all methods and consecutive time points, samples must be from the same starting point pool of cells. For recommendations on which kit to use, refer to the Table of materials.

  1. Preparation and Isolation of Ribosome Protected Fragments (RPFs):
    1. Use appropriate amounts of cells for each point in time (see Table 2 for recommended T cell counts). Treat adherent cells with cycloheximide as described in the manufacturer's protocol.
      Warning: Cycloheximide is highly toxic and can cause mutations. Avoid skin contact and inhalation.
    2. Collect and pool non- or semi-adherent cells from each point in a polypropylene tube and adjust the final concentration, 1 x 106 Cells per mL of cell-specific medium (z. B.supplements RPMI for T cells). Cycloheximide at a final concentration of 0.1 mg / mL, mix by inverting the polypropylene tube, and 1 min.Incubate centrifuge cells for 5 min at 330 x g at 4 ° C. Aspirate medium and wash cells with at least 10 mL of PBS supplemented with cycloheximide (final concentration of 0.1 mg / mL).
    3. Centrifuge cells for 5 min at 330 x g at 4 ° C. Aspirate medium and 100 µL mammalian cell lysis buffer per 10 x 106 Cells. Mix by pipetting and expel completely through a sterile 22 25 gauge needle to lyse the cells.
    4. Transfer the cell lysate to a pre-cooled 1.5 mL tube. Incubate 10 min on ice with periodic inversions. Centrifuge for 10 min at 20,000 x g at 4 ° C to clarify the lysate. Transfer the supernatant to a pre-cooled 1.5 mL tube.
    5. Prepare a 1:10 dilution of the lysate with nuclease-free water and record A.260Reading with a spectrophotometer. Use nuclease-free water as a blank and a 1:10 dilution of the mammalian cell lysis buffer as a standard. Calculate the A260/ mL concentration of lysate according to the following equation:
      (A260 Cell lysate-a260 Mammalian Lysis Buffer) x 10 dilution factor = a260- / mL
    6. Make 200 µL aliquots of the lysate on ice and proceed with nuclease treatment.
      Note: Optionally, prepare total RNA from a 100 µL aliquot, add 10 µL 10% SDS and stir. Store at 4 ° C and continue with 4.3.2. Total RNA from 4sU labeled RNA is recommended (see step 3. Total RNA-Seq).
  2. Ribosome footprint
    1. Perform nuclease treatment immediately without freezing the lysate. Add 7.5 units of the nuclease (included in the recommended kit) for each A260 by lysate. For example: 80 A.260/ mL lysate x 0.2 mL x 7.5 U / A260Nuclease lysate = 120 U nuclease.
      Note: Optionally, titrate nuclease for digestion as instructed by the manufacturer.
    2. Incubate the nuclease reaction for 45 min at RT with gentle mixing. Freeze 200 µL aliquots of the lysate with liquid nitrogen and store at -80 ° C or stop the nuclease reaction by adding 15 µL RNase inhibitor to 200 µL aliquots and continue with step 4.3.2.
  3. Purification of RPFs
    1. Thaw a sample of the digested RPFs nuclease and add 15 µL of RNase inhibitor. Keep the samples on ice.
    2. Clean the RPFs according to the manufacturer's protocol (column cleaning is recommended) and measure the RNA concentration on a spectrophotometer.
  4. rRNA depletion
    1. Use 5 µg of the purified RPF for rRNA depletion.
    2. Follow the manufacturer's protocol (level 1-2, primary rRNA depletion) for rRNA depletion. Measure RNA concentration by depleted rRNA RPFs on a spectrophotometer.
  5. Cleaning the RPF page
    1. Employing 500 ng of rRNA depleted RPFs for side purification.
    2. Prepare side purification RNA control, samples and ladder. Mix 5 µL of the RNA control and 5 µL of denaturation gel loading dye into a 0.5 mL microcentrifuge tube. Mix 10 µL of each RPF with 10 µL of gel loaded or denaturation. Prepare a ladder aliquot (4 µL 20/100 ladder, 1 µL nuclease-free water, and 5 µL denaturing gel load dye). Place it between each sample and control to prevent cross-contamination.
    3. Denature samples and ladder by incubation at 95 ° C for 5 min and immediately on ice. Load 20 µL of each sample (optionally load 10 µL and freeze, the remaining samples at 20 ° C) separated by 10 µL prepared ladder on a 12% or 15% urea-polyacrylamide gel. Load 10 µL of RNA control. Run the gel until the bromophenol blue band reaches the bottom of the gel (180 V, ~ 70 min) (Figure 3).
    4. Stain the gel according to the manufacturer's protocol at 4 ° C. Use a dark-field transilluminator that emits blue light to visualize the RNA. Consume gel slices for each sample corresponding to ~ 28 and 30 nt in length. Check RNA for reference and excise it.
      Note: RPFs are barely visible. Consume slices the size indicated by the RNA controller - containing two oligos of 28 and 30 nt in length - even if samples are not visible.
    5. Pierce a hole in the bottom of the 0.5 mL microcentrifuge tube with a sterile 20-gauge needle. Transfer each gel slice into a separate tube and place limited tubes in a 1.5 mL tube. Centrifuge for 2 min at 12,000 x g. Repeat centrifugation if the gel discs are not completely destroyed in the 1.5 mL tube.
    6. Elute the RNA from the disturbed gel slices with 400 µL nuclease-free water, 40 µL ammonium acetate (5 M) and 2 µL SDS (10%) per night at 4 ° C
    7. Transfer the manure to 1.5 mL filter tubes (provided with the recommended kit) with 1 mL pipette tip (wide-bore tip or homemade 1 mL tip with cut end). Centrifuge for 3 min at 2,000 x g to separate eluted RNA from gel slices. Gently pipette aqueous solution into a 1.5 mL tube. Add 2 µL of glycogen (provided with the recommended kit) and 700 µL of 100% isopropanol and store at -20 ° C for at least 1 h.
    8. Centrifuge at 4 ° C for 20 min at 13,000 x g. Discard the supernatant. The pellet with pre-cooled freshly prepared 80% ethanol at 4 ° C for 10 min at 13,000 x g. Discard the supernatant to wash and dry. Resuspend each sample in 20 µL and the RNA control in 8 µL nuclease-free water. Store at -20 ° C if necessary.
  6. Fragmentation, end repair, 3 'adapter ligation, reverse transcription
    1. Perform the procedure as described by the manufacturer's protocol (fragmentation and end repair, 3 'adapter ligation and reverse transcription).
  7. Purification of the cDNA page
    1. Prepare samples, RNA control and ladder aside purification: Mix 10 µL of each sample and RNA control with 10 µL of denaturing gel dye, respectively load. Prepare a ladder aliquot (4 µL 20/100 ladder, 1 µL nuclease-free water, 5 µL denaturation gel load dye). Place it between each sample and control to prevent cross-contamination.
    2. Denature samples and ladder by incubation at 95 ° C for 5 min and immediately on ice. Load 20 µL of each sample (optionally load 10 µL and freeze, the remaining samples at 20 ° C) separated by 10 µL prepared ladder on a 10% polyacrylamide / 7 - 8 M urea / TBE gel. Load 10 µL of RNA control. Run the gel until the bromophenol blue migrates completely out of the gel (180 V, ~ 60 min).
    3. Stain the gel according to the manufacturer's protocol at 4 ° C. Use a dark-field transilluminator that emits blue light to visualize the RNA and excise the gel slices for each sample corresponding to ~ 70-80 nt.
    4. Proceed as described in steps 4.5.5 - 4.5.8 and resuspend each sample in 10 µL nuclease-free water.
  8. cDNA circularization
    1. Prepare enough Circularization master mix for all reactions by combining the following reagents for each sample on ice: 4.0 µL Circularization Reaction Mix, 2.0 µL ATP, and 2.0 µL MnCl22.0 µL ligase.
    2. Add 10 µL of the master mix to each sample. Mix gently and centrifuge briefly. Incubate samples at 60 ° C for 2 h immediately place samples on ice.
  9. PCR amplification
    1. Follow the manufacturer's protocol (step 1-3, PCR amplification) for PCR amplification. Use 4 µL circularized cDNA for amplification with 9 PCR cycles for primary T cells for best results.
    2. Purify libraries and check their size distribution, according to the manufacturer's protocol (Step 4-8, PCR amplification). The expected size of the amplified library is 140-160 bp (see Figure 4).
    3. For sequencing libraries, see the manufacturer's protocol and the sequencing facility for further guidance.

(5) ChIP-Seq

Note: This protocol has been modified by Blecher-Gonen, Et al. 14 refer to their protocol for more information on ChIP-seq. For all methods and consecutive time points, samples must be from the same starting point pool of cells.

  1. Networking and harvesting of cells
    1. Crosslink corresponding number of cells (see Table 2 for recommended T-cell numbers) for each point in time with a final concentration of 1% formaldehyde in a cell-specific medium (z. B.supplements RPMI for T cells) for 10 min at room temperature with gentle rocking. Stop the crosslinking reaction by adding glycine, a final concentration of 0.125 M.
    2. Centrifuge cells at 330 x g for 5 min at 4 ° C. Discard the supernatant and wash the cells in ice-cold PBS. Repeat step 5.1.2 twice and freeze cell pellets at -80 ° C. Frozen pellets can be stored for at least 6 months.
  2. Cell lysis and sonication
    Note: For all cell lysis and sonication steps, samples should be stored on ice or at 4 ° C to minimize crosslink reversal and protein degradation.
    1. Resuspend cell pellets in 1 mL of ice-cold cell lysis buffer with freshly added protease inhibitors to isolate the nuclei (optionally add phosphatase inhibitors). Incubate 10 min on ice and centrifuge at 2,600 x g for 5 min at 4 ° C
    2. Aspirate the supernatant and resuspend the kernels pellet in 1 mL of ice-cold kernels lysis buffer with freshly added protease inhibitors (optional: add phosphatase inhibitors). Incubate on ice for 10 min. Sonicate the cells to generate a mean DNA size fraction of 0.2-1.0 kb (see Figure 5).
      Note: Sonication conditions must be optimized, depending on the cell type and other conditions (e.g., Cell number, volume and buffer). For primary T cells, recommends sonication for 20-25 cycles (see Materials for detailed description).
    3. Take a 20-50 µL aliquot of sheared chromatin and heat for 10 min at 95 ° C and shake 1,000 rpm to fast rewind crosslink and check chromatin size. Add 2 to 5 µL proteinase K and incubate for 20 min at 56 ° C and 1,000 rpm shaking. Perform heat inactivation for 10 min at 95 ° C and shaking at 1,000 rpm. Purify chromatin with a suitable kit (see Table of materials). Check chromatin size on a 1% agarose gel and use 100 bp plus markers too.
    4. Centrifuge sheared chromatin with a mean DNA size fraction of 0.2-1.0 kb for 10 min at 20,000 x g and 4 ° C to pellet insoluble chromatin and debris. Transfer the supernatant to a new tube and keep on the ice.
    5. Keep approximately 5-10% of the sonicated chromatin as input. Freeze at -20 ° C (used in step 5.5.2).
  3. Pair antibodies to pearls
    1. Pair of 10 µg antibodies (z. B.Anti-RNA Pol II; Anti-histone H3K36me3) in 220 µL PBS (with 0.5% BSA and 0.5% Tween 20) to 80 µL superparamagnetic beads connected to G protein (see Table of materials) for at least 1 h at room temperature with rotation.
    2. Place the pipes on a magnet. Wait until all of the beads are attached to the magnet and remove the supernatant. Another block with 6 µL of sonorized salmon sperm cell DNA with PBS buffer (with 0.5% BSA and 0.5% Tween 20) for 30 min at room temperature with rotation.
    3. Place the pipes on a magnet. Wait until all of the beads are attached to the magnet and remove the clear excess. Wash beads with ChIP IP buffer three times.
  4. Chromatin Immunoprecipitation
    1. Dilute chromatin to 1 mL total volume in Kernel Lysis Buffer with freshly added protease inhibitors (optionally add phosphatase inhibitors). ChIP-IP buffer with freshly added protease inhibitors (optionally add phosphatase inhibitors) to a final volume of 3 mL. Keep on the ice or at 4 ° C while antibody is attached to the beads.
    2. Add diluted chromatin to the antibody-coupled beads from step 5.3.3 and incubate overnight at 4 ° C with gentle rotation.
    3. Wash with the following buffer (1 mL each see Table of Materials) at room temperature for 5 min with rotation, put the tubes back on the magnet and remove the supernatant: Wash Buffer I, wash Buffer II, wash Buffer III and 2 x TE pH 8.0 respectively.
    4. Discard the supernatant and air dry for ~ 5 min.
  5. Reverse networking
    1. Remove samples from the magnets. Pipette 50 µL of Elution Buffer and mix to elute protein-DNA complexes from beads.
    2. Include entering samples from this step forward. Elution buffer for input sample (s) for a final volume of 50 µL (to keep the buffer composition similar to the ChIP samples) and together with the ChIP samples.
    3. Mix 3 µL Elution Buffer and 2 µL RNase (DNase free). Add 5 µL of the mixture to each sample and incubate for 30 min at 37 ° C
    4. Mix 2.5 µL proteinase K, 1 µL glycogen and 1.5 µL elution buffer per sample. Each sample (1 U proteinase K and 20 µg glycogen per sample) add 5 µL of the mixture and incubate for 2 h at 37 ° C
    5. Incubate the samples at 65 ° C overnight (at least 4 h) with shaking to perform reverse cross-linking.
    6. Place the tubes on the magnet for at least 30 s and transfer the supernatant to a new tube. Samples can be frozen at -20 ° C for up to 12 months.
  6. DNA purification
    1. Add 140 µL of well-dispersed paramagnetic beads to 60 µL of the sample (2.3: 1 ratio). Carefully mix the pipette up and down 25 times thoroughly. Make sure that the liquid in each tube is homogeneous. Incubation at room temperature for 2 min.Place the tubes on the magnet for 4 minutes, or until all beads are down, discard the magnet and the supernatant.
    2. Leave the tubes on the magnet and add 200 µL of freshly made 70% ethanol. Incubate the tubes for 30 s without disturbing the beads. Discard the supernatant and repeat this step again. Aspirate the ethanol completely and let the paramagnetic beads air dry for 4 minutes.
      Note: Incomplete ethanol removal can seriously reduce DNA recovery and yield. Dry the pellet just until it is dry. Drying over the pellet can allow DNA recovery and yield.
    3. Remove the tubes from the magnet, and add 20 µL of 10 mM Tris-HCl (pH 8.0). Pipette carefully mix the entire volume up and down 25 times thoroughly. Incubate for 2 min at room temperature. Put the tubes back on the magnet for 4 min and transfer the supernatant to another tube.
    4. Measure the amount of DNA with a suitable fluorometer (see Table of materials).
    5. Make sure that the ChIP of qPCR (diluted 1 µL in 100 µl H2O and use 2 to 5 µL for qPCR) was successful. Use specific primers for a positive (known binding site of the protein of interest) and negative control (z.B. a gene that is breastfeeding and / or not a target of the protein of interest).
      NOTE: Library preparation can be done with 2 ng DNA ChIP depending on the kit (see Table of materials for a suggestion of which kit to use).

Representative Results

4sU labeling: check optimal4sU labeling conditions (cytoplasmic stress, apoptosis, nuclear stress), time and concentration: High levels of 4sU can inhibit the production and processing of rRNA and cytoplasmic as well as nuclear stress30to induce. Therefore, the cells of interest should be tested for 4sU-induced stress as well as apoptosis. Western blot analysis is recommended for visualization of p53 accumulation which indicates nuclear exposure, phospho-EIF2a elevation in cytoplasmic stress, and fluorescence activated cell sorting (FACS) analysis indicated for apoptosis. High and long exposure to 4sU or drugs like thapsigargin or arsenate can cause cellular stress. To induce apoptosis or cell death, cells were treated with BH3I-1 (500 ng / µL) or incubated for 5 min at 95 ° C (heat shock). Annexin V / 7-AAD staining was used to determine apoptosis (Annexin V) and dead (7-AAD) cells. Identification of in-vitro generated primary Th1 cells for 0.5 h with 500 µM 4sU (final concentration) or 1 h with 200 µM 4sU neither signs of cellular stress induced or apoptosis (illustration 1) but lead to sufficient 4sU recording.

RNA labeling times are shortened (≤5 min) which leads to an increase in short-lived intronic sequences compared to longer labeling times. In order to make co-transcriptional splicing prices visible, 4sU marking times should not exceed 30 min. Further details on 4sUlabeling can be found under Rädle Et al. 19

Quality control: The integrity of the RNA is of great importance in the processing of RNA. It is most convenient to test the RNA labeled 4sU RNA after biotinylation by an electrophoretical analysis quality check (see Table of materials). Consider the isolated RNA from step 2.2.2 to check, especially if it is for sequencing of whole RNA. RNA integrity number (RIN) should be ≥8, integrity of the RNA for further processing (Figure 3).

Electrophoretical analysis can also be used to check newly transcribed RNA.Note that newly transcribed RNA contains significantly fewer mature rRNAs compared to total RNA with the typical rRNA bands much less prominent.

Ribosome profiling: PCR amplification of the cDNA library: cDNA amplification (step 4.9.1) is an important step to ensure good sequencing results. Analyze amplified libraries through an electrophoretical analysis. A good example of reinforced libraries shows a peak around 140-160 bp (Fig. 4A). Excessive amounts of adapter, the dimers should be avoided (Figure 4 b) and the samples should be further cleaned using the cleaning procedure page according to the manufacturer's protocol (page Cleaning the PCR products). Too much template or too many PCR cycles result in over-amplification characterized by the appearance of higher than expected molecular weight bands, smeared PCR products and adapter dimer products (Figure 4). For most samples, 1 to 5 µL of circularized cDNA and 9 PCR cycles for amplification usually result in sufficient quantities of the correct PCR product.

ChIP: Chromatin Scissors: Schur optimal conditions must be adapted for each type of cell. Determine scissors conditions (z.B. Number of cycles, high or low power) in advance. Use the same number of cells and the same volume for test purposes, since a lower cell density increases the Schur efficiency. Try to avoid over- or under-cutting the chromatin. Large chromatin fragments can dramatically affect ChIP results by clogging, and shearing epitopes on the protein of interest, which can lead to a lower binding efficiency through which the antibody can destroy. In this experiment, the best results were obtained when the main fraction of the chromatin sheared was around 1,000 bp or slightly lower (Fig. 5A).

Checking the ChIP of qPCR: Before starting the chip, it is advisable to test whether the antibodies used are suitable for ChIP (if possible, use ChIP grade antibodies) by ChIP-qPCR. Check the ChIP for sequencing qPCR before beginning library preparation (see step 5.6.5). Design primers that bind to a known target location of the protein of interest. If the exact target site within a gene is unknown, multiple primer pairs that scan genes and associated regulatory elements can be used. For RNAPII-ChIP from Th1 Ifng cellswhich is transcriptionally upregulated after stimulation and Actin Primers can be used as positive controls. Sox9 and insulin serve as a negative control, as these genes are not present in Th1 cells (Fig. 5 b) are expressed. Don't forget to use exon-spanning primers that are normally used for qPCR mRNA. An IgG control element can also be used to detect specificity of the antibodies used. Immunoprecipitated DNA can be measured with a suitable fluorometer (see Table of materials). Amounts of nonspecifically bound DNA by the IgG control should be significantly lower compared to the amount of DNA bound by the antibodies of interest.

Replicated: Evidence of biological importance: It is strongly recommended to conduct the kinetic experiment for all methods starting from the same pool of cells to ensure that cells have the same identity for all untreated and treated samples (Figure 2). Still, it is best to take small aliquots of prime time points for each method, samples, to a biological replication (z. B.by qPCR, FACS analysis). This allows for a rough estimate of whether the treatment was reproducible for both replicas and you could proceed with the sequencing. Validation of the replicas should be performed using laboratory protocol analysis. Reproducibility of the results in terms of the correlation between FPKM values ​​on repetitions can be assessed and with scatter diagrams (Figure 6) visualized.


Figure 1: Review of the optimal 4sU labeling conditions without disruptive cell physiology (figure from Davari Et al. 11)
(A.) Detection of cell apoptosis by FACS analysis: In Vitro generated or Th0 cells were treated with different concentrations of 4sU (given in brackets) for 0.5 h, 1 h and 2 h. BH3I-1 treatment was used to induce apoptosis as determined by Annexin V, while heat shock (5 min at 95 ° C) was used to induce cell death as determined by 7-AAD. (B.) Western blot analysis for p53 treated 4sU and activated T cells: Samples were labeled with 200 μM 4sU for the indicated time of activation, with the exception of the 0.5 h time point, which was labeled with 500 μM 4sU. (C.) Western blot analysis of phospho-EIF2a and total EIF2a in activated Th1 cells with the same labeling as in (B.). Thapsigargin was used as a positive control. Please click here for a larger version of this figure.


Figure 2: Schematic overview of a kinetic installation tracking genome-wide changes
This scheme shows the setup for the combination of 4sU-Seq, total RNA-Seq ribosome profiling and ChIP-Seq to study genome-wide changes in treatment. Pool cells and set aside the required number of cells for untreated control. Treat remaining cells and split for each method and time point. Label untreated / treated cells for 4sU-Seq with 4sU as described. The timing and samples for each method depends on the specific biological issue being examined. Samples for each time point and method and follow the specific part of the protocol. Please click here for a larger version of this figure.


Figure 3: Quality control with the designation of 4sU RNA
Total RNA and biotinylated RNA obtained from activated Th1 cells were analyzed on a bioanalyzer. 18 s rRNA and 28 s rRNA shown and RNA Integrity Number (RIN) is obtained from the instrument to determine the integrity of the RNA. RIN should be ≥8 to ensure RNA integrity. Please click here for a larger version of this figure.


Figure 4: Bioanalyzer Profiles of the Ribosome Profiling Libraries
(A.) A good library: the example shows a peak at the expected size range (140-160 bp) and no further purification is required. (B.) This example shows excessive adapter dimer reinforced product (120 bp) relative to the desired product (140-160 bp). This library requires further cleaning. (C.) an overly amplified sample: molecular weight peaks higher than expected and smeared PCR amplicons are visible (indicated by arrows). Please click here for a larger version of this figure.


Figure 5: Optimal chromatin size after scissoring and checking the ChIP by qPCR
(A.) Agarose gel image shows the optimal fragment size of sheared chromatin from three samples that were sheared and cleaned on a sonicator for 25 cycles as previously described in the protocol. (B.) Q-PCR results of a total RNAPII chip (Anti-RNA Pol II, 8WG16, ab817) is shown as a percentage of the input. IFNG and Actin Primers served as positive while Sox9 and insulin Negative controls (both genes are not expressed in activated Th1 cells). Please click here for a larger version of this figure.


Figure 6: Comparison of biological replicates (Figure by Davari Et al. ( 11)
Representative scatter plot comparison of expression values ​​(FPKM) between replicated newly transcribed (4sU) RNA-4 h after stimulation of the activated Th1 cells. The green line shows equal FPKM values ​​and rank correlation will be in each parcel.

Duration of the inscription (min)Recommended 4sU concentration (µM)
120100 - 200
60200 - 500
15 - 30500 - 1000
<>500 - 2000

Table 1: Recommended 4sU concentrations (from Rädle, Et al. 19)
The range of recommended 4sU concentrations is indicated for different times of day labeling.

methodNumber of cellsRNA amount
4sU lettering≥2 x 107≥60µg
Ribosome profiling≥2 X107
ChIP-seq≥2 x 107 - 3 x 107

Table 2: Required amount of primary T cells
Minimum amount of primary T cells required for each method. Quantities can be smaller when using other cell types.

Discussion

Analysis of the entire process of gene regulation is necessary to fully understand cellular adaptations in response to a particular stimulus or treatment. Combining Total RNA-Seq, 4sU-Seq, Ribosome, Profiling and ChIP-Seq at different points in time lead to a comprehensive analysis of the most important processes of gene regulation over time. A deep understanding of the biological processes is required in order to define the experimental setup and optimal times.

As methods of studying gene regulation improve rapidly, these protocols can be adapted to rapid changes. Nevertheless, they provide the most important methods for studying basic gene regulation mechanisms in any type of cell. Here we discuss some of the pitfalls and facts one needs to consider when using these methods.

Cells: Cells must be highly viable and if primary isolated cells are used, purity of the cell populations must be ensured (z. B.FACS analysis for primary T cells). Even slightly stressed cells can influence the results of these very sensitive sequencing methods and lower the amount of newly transcribed or translated RNA and lead to undesired readouts of the stress response in the sequencing results. The centrifugation speed mentioned in this protocol for pellet cells is optimized for primary T cells. So adjust the speed depending on the cell type.

4sU effects on cell physiology: In addition to the above options for minimal disturbance, check cell physiology for 4sU addition, further or additional analysis can be carried out, especially if cell numbers are limited. Effects on cell proliferation can be tested by checking the doubling time of the cells by simply counting labeled and unlabelled cells. Nucleolus stress induction could also be examined by analyzing the cell morphology via immunofluorescent staining of the nucleolin and nuclei. To further examine the effects of 4sU, altered global gene expression could be measured by correlating, reading graphs of labeled total RNA after unlabelled total RNA.

Cell phone numbers: For in-vitro Generated T cells it is best to start with at least the height of the cells in table 2specified. Choose appropriate numbers per procedure according to the type of cells. Since T cells have less cytoplasm and RNA compared to other cells, smaller amounts of other cells will most likely be sufficient. For ChIP-Seq, cell numbers depend highly on the antibody used and the expression of the protein of interest within the cells. Lies cells can be used for histone or RNAPII ChIP, while cell numbers must be increased when transcription factors are used, especially when they are expressed at low levels.

4sU labeling and RNA biotinylation: When using adherent cells, 4sU labeling can be passed, as by Rädle Et al.described. 19 since cells integrate 4sU very quickly, it can be added directly to the medium of suspension, trailer or semi-adherent cells.

It is recommended to start the biotinylation with 60-80 µg RNA. However, lower amounts of RNA can be used, although we do not test for less than 30 µg. Adding a coprecipitant (e.g.GlycoBlue) as RNA precipitates if the pellet is difficult to see. Duffy Et al. have shown that methylthiosulfonate activates biotin (MTS-biotin) more efficiently with 4sU labeled RNA than HPDP-biotin31responds. Therefore, it may be useful considering the switch to MTS-biotin, especially for the recovery of small RNAs that tend to have less uridine residues (refer to Duffy's biotinylation protocol Et al .; see Purification of 4sU labeled RNA called its Experimental Procedure).

For the recovery of newly transcribed RNA it is possible to use paramagnetic beads or RNA cleanup beads of your choice. Always take into account that these kits may or may not purify for certain RNAs. If you are interested in miRNAs, consider e.g. B. with special kits for miRNA separation and sequencing.

Quantification of the newly transcribed RNA: In order to accurately quantify newly transcribed RNA, measurement can be carried out using a suitable fluorometer (see Table of materials). Within 1 h after exposure for 4 seconds, newly transcribed RNA represents approx. 1-4% of the total RNA. Newly transcribed RNA marked by 1 h, activated T cells consists of approx. 90-94% of the rRNA11.

Ribosome profiling: In establishing the method, it is found that using 1.5 times the amount of nuclease than the original protocol suggested guarantees proper digestion. Also, no adverse effects were reported for increased levels of nuclease. Because it is quite difficult to overdigest the RPFs while they are part of the RNA bound by ribosomal proteins, you can still easily increase the amount one titrate to achieve optimal nuclease digestion.

If less than 500 ng of RPF RNA have been recovered, in step 4.4.2, repeat the rRNA depletion and pool purified RPFs with RNA Clean & Concentrator-5 columns. Alternatively, load two identical samples side by side onto the gel (step 4.5.3) and pool gel slices during RNA elution from the gel (step 4.5.6).

It is recommended that you cut the RPFs on a gel as close as possible to the 28 and 30 nt bands. This helps in eliminating unwanted fragments of rRNAs and tRNAs that are later part of your library and reducing sequencing reads for your RPFs.

It is also recommended to avoid UV light during gel cleaning. This can create nicks in the RNA fragments as well as pyrimidine dimers, which can significantly affect the end of the library preparation and sequencing results.

Library preparation and sequencing of data: Ribosome profiling protocol enables a cDNA library to be generated for sequencing. Samples generated by 4sU labeling can be used directly for library preparation with an appropriate RNA sequencing kit. Since newly transcribed RNA, especially if with short times, labeling is not yet polyadenylated, no poly-A selection should be carried out. Instead, we recommend rRNA depletion to prevent reducing the sequencing depth for the actual sample. With T cells, we started with 400 ng re-transcribed and total RNA (depending on the kit, see Materials), performed rRNA depletion and reduced cycles for PCR amplification to minimize PCR bias. Library preparation can be done with less starting material. To take library complexity into account many PCR cycles should be optimized.

There are also many library preparation kits available for ChIP-Seq. In our hands library preparation also worked starting with 2 ng DNA ChIP (see Materials for a suggestion on which kit to use). Be sure to check the indexes for color balance during sequencing. We recommend a sequencing depth of ≥40 x 106 reads each 4sU-Seq, total RNA-Seq and ChIP-Seq samples and ≥80 x 106 reads samples for ribosome profiling. The sequencing depth depends on the sample and the downstream bioinformatics analysis and should be carefully considered. In order to analyze intronic reads for cotranscriptional splicing, 100 bp paired-end sequencing must be chosen.

Bias sequencing: Sequencing has become the gold standard in determining global changes in transcription, translation and transcription factor binding. In the last few years existing methods had reached their limits or new techniques were developed to carry out smaller and smaller starting amounts of RNA sequencing. This requires amplification of the cDNA, which introduces noise or distortion. Recently, unique molecular identifiers (UMIs) have been developed to experimentally identify duplicates introduced by PCR. Recently, it was shown that UMIs only perform mild sequencing and false discovery rates for differential gene expression32improve. Still, consider adding unique molecular identifiers (UMIs) for all sequencing libraries to control library complexity, especially when starting with low amounts of RNA and when many PCR cycles are required.

Buffers and stock solutions: All buffers for 4sU-Seq and ribosome profiling must be prepared under strict RNase-free with nuclease-free water.It is best to buy pre-made nuclease-free NaCl, Tris-HCl, EDTA, sodium citrate, and water. To ensure nuclease-free conditions, an RNase decontaminating solution can be used to clean pipettes or surfaces. All buffers for ChIP-Seq must be at least DNase-free and can be stored at room temperature. Always add protease inhibitors and optionally phosphatase inhibitors just before use and on ice.

Bioinformatics: Analysis of all sequencing data (d. H., ChIP-Seq, RNA-Seq and Ribsosome profiling) includes quality control (e.g.with FastQC, http://www.bioinformatics.babraham.ac.uk/projects/fastqc/), trim adapter (z. B.with cutadapt20) followed by mapping to the reference genome for the cells examined. For RNA-Seq data (total and 4sU-Seq) and ribosome profiling data, a spliced ​​RNA-Seq mapper is required, e.g. B. ContextMap 221. For ChIP-Seq data, unspliced ​​axes are sufficient with BWA-MEM-22 . Gene expression can be calculated using the RPKM model (read per kilobase exon per million fragments mapped)1, after determining counts per gene with a program, z. B. FeatureCounts23to read. For the summit call of ChIP-Seq data, there are a number of programs, e.g., MACS24 or GEM25. Subsequent analyzes in R26, especially using Bioconductor Project27tools provided.

Here is a major challenge in integrating 4sU and total RNA levels and normalizing translational activity from ribosome profiling. A classic approach to solving this problem is to normalize genes to a level of housekeeping. By reducing random fluctuations for individual housekeeping genes, it is not recommended to just add a few housekeeping genes median levels for a larger group, z. B. to use, but the> 3,000 housekeeping genes compiled by Eisenberg and Levanon28 . For the calculation of the RNA turnover rates of translations of 4sU total RNA, normalization based on mean turnover rates (e.g., provided an RNA half-life of 5 h)29. Since this does not assume any general changes for housekeeping genes, we recommend however using analysis approaches independent of normalization, e.g.Correlation based clusters of a time series of different data types to identify groups of genes with different behavior in transcription and translation during activation. For a detailed description of laboratory protocols integration of the various data types, we refer to the original publication11.

Analysis of sales prices and data integration: A recently published paper33 Half-lives determined by a bundled gene (MGC) compared to global methods could show that the half-lives correlated best with those obtained by metabolic labeling methods compared to other methods (e.g., General inhibition of transcription by drugs). However, it should be noted that differences between half-life calculations can arise and are described 15,34been. We make up most of the problems and differences introduced by the stress response due to prolonged 4sU exposure. Hence, it is imperative to rule out the stress response introduced by 4sU labeling. To check further conversion rates, we recommend using MGCs.

In addition, an integrative data analysis (z.B. Regulation of long non-coding RNAs)35,36be used.

Disclosures

The authors state that they have no financial conflicts of interest.

Acknowledgments

We thank Lars Dölken for advice on establishing 4sU labeling for primary T cells; Elisabeth Graf and Thomas Schwarzmayr for critical help with library generations and sequencing; Dirk Eick and Andrew Flatley for providing RNAPII and T cell antibodies; N. Henriette Uhlenhaut and Franziska Greulich for help in preparing the library for ChIP-Seq; Caroline C. Friedel was supported by grants FR2938 / 7-1 and CRC 1123 (Z2) from the German Research Foundation (DFG). Elke Glasmacher was supported by the grant GL 870 / 1-1 from the German Research Foundation (DFG) and the German Center for Diabetes Research (DZD), Helmholtz Center Munich.

Materials

SurnameCompanyCatalog NumberComments
4sU labeling
4-thiouridine (100 mg)Carbosynth13957-31-8Prepare 50 mM stock in sterile H2O / PBS; store at -20 ° C in aliquots of 50-500 µl; do not refreeze.
1.5 ml safe-lock tubesEppendorf30121589Optional
1.5 ml screw-top polypropylene tubesSarstedt72692005Compatible with dimethylformamide
15 ml tubesBD Falcon352096Compatible with dimethylformamide
2.0 ml screw-top polypropylene tubesSarstedt72694005Compatible with dimethylformamide
50 ml tubesBD Falcon352070Compatible with dimethylformamide
Agencourt RNAClean XPBeckman CoulterA63987We recommend to use these paramagnetic beads. Aliquot and store at 4 ° C
chloroformSigma Aldirch372978WARNING - HAZARDOUS TO HEALTH
DimethylformamidesSigma AldrichD4551
Dithiothreitol (DTT)Roth6908.1Prepare as 100 mM DTT in nuclease-free H2O; always prepare fresh
EthanolMerck1.00983.1000
EZ-Link Biotin-HPDP (50 mg)Pierce21341Prepare 1 mg / ml stock solution by dissolving 50 mg biotin-HPDP in 50 ml DMF. Gentle warming enhances solubilization. Store at 4 ° C in aliquots of 1 ml. DMF dissolves some plastic materials. We recommend to use glass pipettes to transfer DMF from ist stock glass bottle to 50 ml Falcon tubes.
High sensitivity DNA kitAgilent Technologies5067-4626
IsopropanolMerck1.09634.1011
NaCl (5M)Sigma Aldrich71386Stock solution
nuclease-free EDTA (500 mM), pH 8.0Invitrogen15575-020Stock solution
Nuclease-free H.2OSigma AldrichW4502Stock solution
nuclease-free Tris Cl (1M), pH 7.4Lonza51237Stock solution
Phase Lock Gel Heavy tubes (2.0 ml)5Prime2302830Use in step 1.3.4.
Polypropylene 15 ml centrifuge tubesGreiner Bio-One188271Or equivalent; they have to tolerate up to 15,000 × g
QIAzol Lysis Reagent (200 ml)Qiagen79306Use this or equivalent TRI reagent for RNA isolation, WARNING - CORROSIVE and HAZARDOUS TO HEALTH! Ensure immediate access to phenol antidote (PEG-methanol)
Qubit RNA HS assay kitLife TechnologiesQ32852Use this kit for quantifying RNA quantity in step 1.4.11
RNeasy MinElute KitQiagen74204Optional; includes Buffer RLT
Sodium citrateSigma AldrichC8532Prepare 1.6 M stock solution using nuclease-free water
Tween 20Sigma AldrichP1379
µMacs Streptavidin KitMiltenyi130-074-101Store at 4 ° C, includes µMacs columns used in step 1.4.6. (store at RT)
Cell viability and stress assay
PE Annexin V Apoptosis Detection Kit IBD Biosciences559763Optional
ThapsigarginSigma-AldrichT9033Optional
p53abcamfrom26Optional
p-EIF2a (Ser51)Cell signaling9721Optional
BH3I-1Sigma-AldrichB 8809Optional
Buffers
4sU washing bufferstore at RT100 mM Tris pH 7.4, 10 mM EDTA, 1 M NaCl, 0.1% Tween 20 in nuclease-free H2O
Biotinylation Buffer (10x)store at 4 ° C100 mM Tris pH 7.4, 10 mM EDTA in nuclease-free water; make aliquots of 1 ml; store at 4 ° C
RNA precipitation bufferstore at RT1.2 M NaCl, 0.8 M sodium citrate in nuclease free water. Prepare in advance under nuclease-free conditions. Store at room temperature in 50 ml falcon tubes.
Equipment
2100 bioanalyzer instrumentAgilentG2939BA
RNA 6000 Nano KitAgilent5067-1511Use this kit to verify RNA integrity in step 1.3.10
RNA 6000 Pico KitAgilent5067-1513Optional
UV / VIS spectrophotometerThermo ScientificNanoDrop 1000Or equivalent. Use in step 1.2.2 / 3.1.8 / 3.3.3 / 3.4.3
High-speed centrifugeThermo ScientificHeraeus Multifuge X3ROr equivalent equipment capable of reaching 13,000 × g
High-speed rotorThermo ScientificFiberlite F15-6 x 100y
Adapters for 15 ml tubes
Refrigerated table-top centrifugeEppendorf5430 ROr equivalent.
ThermomixerEppendorfThermomixer COr equivalent.
Magnetic standMiltenyi Biotec130-042-109One stand holds 8 µMacs columns.
Ultra-fine scaleMettler ToledoML204TOr equivalent.
E-Gel iBase Power SystemInvitrogenG6400UKFor RNA gels; or equivalent.
E-Gel EX 1% agarose precast gelsInvitrogenG4020-01For RNA gels; or equivalent.
DynaMag-2 Magnet-1 eachLife Technologies12321D
RNaseZapSigmaR2020Optional
TruSeq stranded total RNA library prep kitIlluminaRS-122-2201Or equivalent. For T cells we used 400 ng 4sU and Total RNA with 11 cycles for PCR amplification. rRNA depletion is included in this kit
NanodropThermo Scientificuse a Nanodrop or equivalent instrument to measure RNA concentration
Ribosome Profiling
TruSeq Ribo Profile kit (Mammalian or Yeast)IlluminaRPYSC12116 (Yeast)
TruSeq Ribo Profile kit (Mammalian or Yeast)IlluminaRPHMR12126 (Mammalian)
Illustra MicroSpin S-400 HR ColumnsGE Healthcare27-5140-01
RNA Clean & Concentrator-25 kitZymo ResearchR1017
RNA Clean & Concentrator-5 kitZymo ResearchR1015
Ribo-Zero Gold rRNA Removal Kit (Human / Mouse / Rat)IlluminaMRZG126 or MRZG12324
(High Sensitivity DNA Kit)Agilent Technologies5067-4626Already needed for 4sU-seq
All other consumables and equipment are listed in the user guide!!!Carefully read the user guide and order required consumables in advance (consider a long delivery time for some consumables e.g. gels)
Chip
10 mM Tris-HCl (pH 8.0)gereral lab supplier
100 bp plus markersThermo FisherSM0323
16% formaldehydeThermo Fisher28908Add to a final concentration of 1%
70% EtOHgereral lab supplierAlways prepare fresh
Agarosegereral lab supplier
Agencourt RNAClean XP beadsBeckman CoulterA63987We recommend to use these paramagnetic beads. Aliquot and store at 4 ° C
ChIP library preparation kitKapaBiosystemsKK8504Or use the kit of your choice
DNA low bind microcentrifuge tubesEppendorfZ666548-250EAor equivalent
Dynabeads Protein GInvitrogen10004DUse these superparamagnetic beads coupled to protein G in step 4.3.1 .; Bring to RT before use
Glycinegereral lab supplierPrepare a 2M stock solution
GlycogenRoche10-901-393-001
MinElute PCR Purification KitQiagen28004Use this kit (or equivalent) to purify chromatin in step 4.2.4.
Phosphatase inhibitor (PhosStop)Roche4906837001Add freshly to the buffer and keep on ice
Power SYBRgreen Master mixThermo Fisher4367659
Protease Inhibitor (cOmplete, EDTA-free)Roche11873580001Add freshly to the buffer and keep on ice
Proteinase KInvitrogen25530049
Qubit dsDNA HS Assay kitInvitrogenQ32851Use this kit for quantifying DNA quantity in step 4.6.4. on a qubit fluorometer
Rnase, DNase freeRoche11-119915001
Salmon sperm (sonicated to around 100bp)SigmaD1626
TE pH 8.0gereral lab supplier
Antibodies (ChIP grade if possible)
anti-RNA Pol II [8WG16]abcamfrom817
anti-histone H3K36me3abcamfrom9050
or antibody of interest
Buffers
Binding / blocking bufferStore at RTPBS with 0.5% BSA and 0.5% Tween 20
Cell-Lysis bufferStore at RT5 mM pipes [pH 8.0], 85 mM KCl, and 0.5% NP40
ChIP IP bufferStore at RT0.01% SDS; 1. 1% Triton X-100; 1.2 mM EDTA; 16.7 mM Tris-HCl, pH 8.1; 16.7 mM NaCl
Elution bufferStore at RT up to 6 months10 mM Tris-HCl (pH 8.0), 5 mM EDTA (pH 8.0), 300 mM NaCl and 0.5% SDS
Nuclei-Lysis bufferStore at RT50 mM Tris [pH 8.0], 10 mM EDTA, and 1% SDS
Wash buffer I.Store at RT0.1% SDS; 1% Triton X-100; 2 mM EDTA; 20mM Tris-HCL pH 8.1; 150 mM NaCl
Wash buffer IIStore at RT0.1% SDS; 1% Triton X-100; 2 mM EDTA; 20 mM Tris-HCl pH 8.1; 500 mM NaCl
Wash buffer IIIStore at RT0.25 M LiCl; 1% NP-40; 1mM EDTA; 10 mM Tris-HCl, pH 8.1
Equipment
2100 bioanalyzer instrumentAgilentG2939BAuse this instrument for electrophoretical analysis
NanodropThermo Scientific
Bioruptor TBX microtubes 1.5 mlDiagenodeC30010010
or tubes special for your sonication device
Bioruptor sonication device or sonication device of your choiceSonication of T cells with Bioruptor: 20 - 25 cycles (30 s on, 30 s off at high in two 1.5 ml bioruptor microtubes with 500 µl each tube)
Magnetic stand for tubes
Thermomixer
Agarose gel electrophoresis
Qubit fluorometerThermo ScientificUse this fluorometer for quantifying low amounts of RNA / DNA

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References

  1. Mortazavi, A., Williams, B. A., McCue, K., Schaeffer, L., Wold, B. Mapping and quantifying mammalian transcriptomes by RNA-Seq.Nat Methods. 5, (7), 621-628 (2008).