GSM8012628: ESC H3K36me3 sgBrd8; Mus musculus; OTHER
Briefly, 1 × 105 cells were washed twice with wash buffer (20 mM Tris-HCl pH 7.4, 150 mM NaCl, 0.5 mM spermidine and 1× protease inhibitors). Then cells were bound to concanavalin A beads (BP531, Bangs Laboratories) in binding buffer (20 mM Tris-HCl pH 7.4, 10 mM KCl, 1 mM CaCl2 and 1mM MnCl2). The bead-bound cells were washed once with buffer containing 0.01% digitonin, and primary antibodies were added to bead-bound cells in antibody buffer (4 mM EDTA and 0.2% BSA in wash buffer containing 0.01% digitonin) and rotated overnight at 4 °C. The primary antibodies below were used: anti-BRD8 (ab17969, Abcam), anti-IGF2BP2 (11601-1-AP, Proteintech), anti-H3K27me3 (07-449, Millipore), anti-H3K4me1 (ab176877, Abcam), anti-H3K4me3 (ab8580, Abcam), anti-H3K27ac (ab4279, Abcam) and anti-H3K9me3 (ab8898, Abcam). Secondary antibody (ABIN101961, Antibodies-Online) was added to bead-cells-antibody in wash buffer and further incubated at RT for 1 h. pG-Tn5 (S603-02, Vazyme) was add to cells in '300-wash' buffer (20 mM Tris-HCl pH 7.4, 300 mM NaCl, 0.5 mM spermidine and 1× protease inhibitors, 0.01% digitonin) at RT for 1h. Tn5 was activated with 300-wash buffer supplemented with 10 mM MgCl2 and washed five times with 500 μl 300-wash buffer. To stop the Tn5 reaction, 10 μl of 0.5 M EDTA, 3 μl of 10% SDS and 1.5 μl of proteinase K (20 mg/ml) were added 55 °C for 1 h. DNA fragments were extracted and PCR amplified using NEBNext HiFi 2× PCR Master mix (M0541L, NEB) using the settings: 72°C, 5 min, 98°C 30 s and 12-14 cycles of: 98°C 10 s, 63°C 10 s and 72°C 30 s. 300-500 bp DNA fragments were purified with the VAHTS DNA Clean Beads (N411-02, Vazyme). Libraries were sequenced by an Illumina sequencer. Libraries were prepared using NEBNext® Ultra™ II DNA Library Prep Kit for Illumina