RELACS: a novel in-nuclei barcoding strategy for high-throughput ChIP-seq

Chromatin immunoprecipitation followed by deep sequencing (ChIP-seq) is a widely used technique to study the genome-wide distribution of chromatin-associated proteins. Despite many improvements, ChIP-seq still remains a labor-intense process for which sample preparation (chromatin extraction, fragmentation, immunoprecipitation and library preparation) is performed individually for each sample. Here we present a novel in-nuclei chromatin barcoding strategy for high-throughput ChIP-seq. The method, called RELACS (Restriction Enzyme-based Labeling of Chromatin in Situ) relies on intra-nuclear chromatin fragmentation using restriction enzymes and ligation of DNA barcodes to chromatin. Nuclei labelled with different barcodes are pooled for combined ChIP ensuring maximal data comparability and throughput. RELACS has been designed as a broadly applicable method for fixed cells extracted from any tissues, and demonstrated on active and repressive histone modifications as well as transcription factors.


Reagents
• Fixation buffer: dilute formaldehyde at 1% final concentration in serum-free cell culture media such as D-MEM or other suitable media. The solution must be prepared just before use. Use a fresh sealed formaldehyde ampule every time (16% Formaldehyde, Thermo Scientific, cat. No. 28906).
• Protease inhibitor cocktail (PIC), 100X: dissolve one tablet of Complete EDTA-free (Roche, cat. No. 11873580001) in 500 µl of molecular biology grade water. Aliquot and store the resuspended tablets at -20 °C for up to three months or at 4 °C for a maximum of two weeks.
Store the prepared solution at 4 °C (up to one month). Supplement with 1X PIC just before use.
Store the prepared solution at room temperature.
Store the prepared solution at 4 °C. Equilibrate the solution at room temperature before use to dissolve SDS precipitates. Supplement with 1X PIC just before use.
• iDeal ChIP-seq kit for histones, reagents (Diagenode, catalog no. C01010173): 5X buffer iC1, wash buffers 1-4, ChIP elution buffer • Dynabeads (Invitrogen) (A or G type depending on the antibody) • Antibody of interest: use ChIP-seq grade antibodies whenever possible. Based on the antibody quality the amount of antibody per IP can vary greatly. An excess of antibody may lead to increased background. Although not crucial for success of a ChIP-seq experiment, pilot tests might be needed.
Centrifuge at 300 g for 5-10 mins at room temperature. Remove the media.

5.
Mix by inverting the tube and centrifuge at 300 g at room temperature for 5 mins. Do not incubate to prevent over-fixation. CRITICAL: Avoid prolonging centrifugation time since glycine is not sufficient to fully block the formaldehyde.
CRITICAL: In case of sticky cells or low cell numbers the addition of 1 mg/ml BSA (or 10% (vol/vol) serum) and 10 mM EDTA to the suspension may help to prevent stickiness and to increase recovery.

6.
Remove the supernatant by pipetting and discard appropriately.

7.
Resuspend cells in PBS and centrifuge at 300 g at room temperature for 5-10 mins.
Discard the supernatant by pipetting.

8.
Repeat the wash step using PBS supplemented with 1X protease inhibitor cocktail. If required, aliquot the suspension into tubes of suitable volume before centrifuging.
For very low cell numbers this step may be omitted.

C -Whole tissues
1. Cut the tissue into small pieces (approx. 1 cubic millimeter) into a Petri dish containing some cell culture D-MEM media.
Collect the tissue using a 1 ml tip with cut tip and transfer into a Dounce homogenizer (loose pestle, type A). Decant and remove the excess of media.

3.
Add Fixation buffer and start homogenizing the tissue with a couple of strokes (2-3 max). Start the timer set at 15 minutes.

4.
Filter the preparation through a 70 µm nylon cell strainer (Falcon, 352350) over a Falcon tube. Wash the strainer using fixation buffer. Volumes used are not critical, but for convenience do not exceed the 4-6 ml of total volume.

5.
When the incubation time with fixative is finished, add 125 mM glycine final concentration (110 µl of a 1.25 M glycine solution per ml of suspension; measure suspension volume using a graduate tip during waiting times).

6.
Mix by inverting the tubes and centrifuge at 500 g at room temperature for 5 mins.
Do not incubate to prevent over-fixation.

7.
Remove the supernatant by pipetting and discard appropriately.

8.
Resuspend cells in PBS and centrifuge at 500 g at room temperature for 5 mins.
Discard the supernatant by pipetting. CRITICAL: avoid processing a large number of tubes at the same time since cell pellets can easily detach at this step.
Centrifugation time or speed might also be increased.

9.
Repeat the wash step using PBS supplemented with 1X protease inhibitor cocktail.
For very low cell numbers this step may be omitted.
CRITICAL: pellets can be stored at -80 °C for up to a year or more. Do not store fixed cell pellets at -20°C since it can compromise sample quality. [PAUSE POINT]

1.
Fully resuspend formaldehyde-fixed cells with 1 ml of Lysis buffer supplemented with Protease Inhibitor Cocktail (10 µl of a 100X solution). Use a pipette and do not vortex. • For cell numbers between 10,000 to 100,000, resuspend in 100 µl volume.
• Up to about 5 million of cells can be treated using these parameters.
• Quality control 1 (Figure 1a): Check the cell suspension on a phase contrast microscope (use 4 µl of nuclei preparation), or by DAPI staining (add directly on a microscope slide 4 µl of nuclei preparation plus 4 µl of DAPI working solution). In this step one can evaluate nuclei shape in a respective cell type, cell integrity and cell density. Insert the tube into the Covaris adaptor.

4.
Extract nuclei by sonication using the NEXSON procedure until nuclei isolation is satisfactory: • Sonicate the sample for 15-30 seconds.
CRITICAL: some cell types sediment fast in the Covaris tube. Mix by inversion just before starting sonication to ensure the cells are fully resuspended. AFA fiber does not mix sufficiently the sample when the instrument is set at that power.
• Quality control 2: Check nuclei extraction on a phase contrast microscope (use 4 µl of nuclei preparation), or by DAPI staining (add directly on a microscope slide 4 µl of nuclei preparation plus 4 µl of DAPI working solution).
• If the nuclei isolation is not satisfactory, treatment time can be prolonged up to 5-8 minutes.
CRITICAL: proceed step by step and check the nuclei preparation under the microscope. Nuclei are very robust, but too much sonication leads to the breakage of nuclei and reduced chromatin recovery.
We suggest checking nuclei isolation at every 1-2 minutes of treatment. Alternatively, if the cells are very resilient, nuclei isolation control can be done after longer treatment times.
• Stop the nuclei extraction when it is satisfactory (about 70% of isolated nuclei). Figure 1b shows a 8 preparation of well-isolated nuclei.

5.
Transfer the nuclei preparation into a 1.5 ml Eppendorf tube (not Lo-bind). In case of high cell numbers, split the sample: the prepared pellets should not contain more than about 5 million cells.

7.
Gently resuspend the pellet in 50 μl of 0.5% SDS and incubate at room temperature for 10 minutes. • In this step the SDS will be quenched and the nuclei will return to the original size ( Figure 1d,

9.
Quality control 5 (optional): quantification of nuclei numbers (optional but suggested for cell numbers higher than 500,000). Store nuclei in the digestion mix at 4 °C till the final results of quantification of cell numbers. • Take 5% of the nuclei in CutSmart buffer (12.5 µl) and resuspend them 88 µl of ChIP elution buffer.
• Transfer the preparation into a Covaris MicroTube and sonicate 5 minutes at Peak power: 105W, • The optimal size distribution is shown in Figure 2

CRITICAL: Normalize nuclei between samples resuspending them in EB (Qiagen), so
that the nuclei density is between 10,000 to 500,000 nuclei per 25 µl EB. The samples to be pooled together after barcoding should have very similar densities to avoid strong differences in sequencing depth. • The optimal cell density is 100 to 500,000 cells per barcoding reaction. The procedure was tested up to one million cells, showing a slight reduction in performance.

18.
Resuspend nuclei into the calculated EB volume.

26.
After ligation and prior pooling, inhibit ligase by adding 300 mM NaCl final concentration in each well (~5 µl of a 3M NaCl solution).

27.
Pool all the barcoded nuclei in a 1.5 ml Eppendorf tube. • Optional: for very low cell numbers (< 100,000 total in the pool) add 1X BSA final concentration to prevent stickiness of cells to the tubes.

28.
Pellet down the nuclei (5000 g, 10 min, 20 °C) and remove the supernatant (bubbles first to prevent pellet detachment). • CRITICAL: The pellet is very flimsy attached to the bottom of the tube and can get easily lost.
• The pellet should be visible and white, also when using 1,000 cells.
• Optional: save the supernatant and and re-centrifuge again (11000 g, 5 min, 20 °C) to collect any leftover pellet.

Appendix I -Procedure to anneal barcoded adaptors
• Resuspend oligos in annealing buffer to the concentration of 100 µM.
• Make a dilution of the oligo to 15 µM to the final volume of 100 µl in annealing buffer (15 µl adapters 100 µM + 85 µl annealing buffer). Store the 100 µM unannealed stock oligos at -80 °C • Heat the oligos at 95 °C for 2 min in a thermoblock.
• Switch off the thermoblock and wait that it cools down to room temperature (it takes about 2-3 hours).
• Divide into suitable aliquots and store at -20 °C.
Sequences of barcoded adaptors are provided in the attachments.

Supplementary Files
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