GSM5862770: IPS_GABA_H3K27ac_t90_Rep1; Homo sapiens; OTHER
CUT&RUN was performed as previously described. Nuclei (50,000) were extracted from neurons using NE1 buffer (20 mM HEPES-KOH pH 7.9, 10 mM KCl, 0.5 805 mM Spermidine, 0.1% Triton X-100, 20% Glycerol and Roche Complete Protease Inhibitor EDTA-Free and used as the input source. Nuclei bound to Concanavalin-coated magnetic beads (Bangs Laboratories BP531) were incubated with the relevant antibody: H3K27ac (Abcam AB4729), FOS (Abcam AB208942), rabbit IgG (Abcam AB46540) or guinea pig IgG (Novus NBP1-2763). The next day, the beads were placed on a magnet to remove supernatant, washed, and incubated with Protein AG-MNase. To fragment the DNA, samples were first chilled to 0º C, the digestion was then activated by the addition of 2 mM of CaCl2. Reactions were stopped by 2×Stop buffer (340mM NaCl, 20mM EDTA, 4mM EGTA (Alfa Aesar J60767), 0.05% Digitonin, 100ug/mL RNAse A (Thermo Fisher Scientific EN0531), 50ug/mL Glycogen). DNA fragments released from the nuclei were purified by extraction with phenol-chloroform and ethanol precipitated. DNA was quantified with a fluorescence-based method using Qubit 4 Fluorometer dsDNA HS assay. CUT&RUN DNA (10ng) was processed using NEBNext Ultra II DNA Library Prep Kit (New England Biolabs 816 E7645) for library preparation according to manufacturer's instructions. Briefly, purified DNA was repaired, ligated with 1.5μM NEBNext Adaptors and amplified for 14 cycles by PCR. DNA was enriched for fragments of size 150-350bp for transcription factor and 150-800 bp for histone markers using Ampure XP beads. Final libraries were quantified with Qubit dsDNA HS assay and the size distribution was determined by Agilent 4200 TapeStation. Libraries were mixed at an equal ratio to achieve a final concentration as recommended by the manufacturer and sequenced on an Illumina HiSeq 4000 system.