GSM9008137: ATAC-seq in MCR_S/MCR_S primary cortical neurons untreated 4; Mus musculus; ATAC-seq
CUT&RUN reactions were performed as previously described (Meers et al., 2019), with the addition of 20mM sodium butyrate when profiling BAHCC1, SIN3A, SIN3B and H3K27ac CUT&RUN reactions were performed following the V3 of the protocol (28), with minor modifications. An equal number of cells (up to 350,000 for each sample) was harvested using Trypsin (0.05%) solution and washed once with 1X PBS at room temperature. Cells were then washed at three times at RT in Wash Buffer (20mM HEPES-NaOH pH 7.5, 150mM NaCl, 0.5mM Spermidine supplemented with EDTA-free Protease Inhibitor Cocktail (APExBIO)). Following the third wash, cells were resuspended in 1 mL Wash Buffer and incubated while gently rotating with 20 µL of Concanavalin A-coated magnetic beads (EpiCypher), which had been previously washed, activated and resuspended with Binding Buffer (20mM HEPES-NaOH pH 7.5, 10mM KCl, 1mM CaCl2, 1mM MnCl2). After 10 minutes incubation, the buffer was removed, and the beads were resuspended in 150 µL of Antibody Buffer (Wash Buffer with 0.1% Digitonin (Sigma) and 2mM EDTA pH 8) containing the proper dilution of each antibody of interest: Total Pol2 (Rpb1 CTD (4H8) Mouse mAb #2629), Ser2P and Ser5P (a kind gift from Hiroshi Kimura) . The cells were then incubated with each antibody, with gentle rotation overnight with a 180° angle at 4°C. On the next day, cells were washed twice with ice-cold Dig-Wash Buffer (Wash Buffer with 0.1% Digitonin), resuspended in 150 µL of Dig-Wash Buffer supplemented with 1 µL of custom-made pA/G-MNase every mL and left rotating for 1h at 4°C. After that, cells were washed twice with Dig-Wash Buffer, resuspended in 100 µL of Dig-Wash Buffer, and placed into a thermoblock sitting on ice. To initiate the cleavage reaction, 2 µL of a freshly diluted (from a 1M stock) solution of 100mM CaCl2 was added to each tube, and the tubes were left at 0°C for 30 minutes. To halt the reaction, 100 µL of 2X STOP Buffer (340mM NaCl, 20mM EDTA pH 8, 4mM EGTA pH 8, 0.05% Digitonin, 100 µg/mL RNAse A, 50 µg/mL Glycogen) were added to each tube, and cleaved DNA fragments were released by incubating the samples for 30' at 37°C. The tubes were then centrifuged for 5 minutes at 4°C / 16,000g and the supernatant was collected. Finally, the DNA was purified by standard Phenol/Chloroform extraction using the 5PRIME Phase Lock Gel Heavy tubes (QuantaBio), and the success of the CUT&RUN reaction was assessed by running the positive control (H3K27me3 or H3K4me3) on a Tapestation (Agilent Technologies) using a High Sensitivity D1000 ScreenTape (Agilent Technologies). PolyA selected libraries were generated using the CORALL mRNA-seq V2 kit (Lexogen 177.96) according to the manufacturer's protocol.