GSM5464137: D30_IMQ_PolII_CNR_Rep1; Mus musculus; OTHER
Cells were FACS sorted according to characteristics: FACS markers. 400,000 cells were resuspended in crosslinking buffer (10mM HEPES-NaOH pH 7.5, 100mM NaCl, 1mM EGTA, 1mM EDTA, 1% formaldehyde) and rotated at room temperature for 10 min. Crosslinked cells were quenched with glycine at a final concentration of 0.125M for 5 min at RT. Cells were washed with cold PBS and resuspended in NE1 buffer (20mM HEPES-KOH pH7.9, 10mM KCl, 1mM MgCl2, 1 mM DTT, 0.1% triton X-100 supplemented with Roche complete protease inhibitor EDTA-free) and rotated for 10 min at 4'C. Nuclei were washed twice with CUT&RUN wash buffer (20mM HEPES pH7.5, 150mM NaCl, 0.5% BSA, 0.5mM spermidine supplemented with protease inhibitor) and incubated with concanavalin-A (ConA) beads washed with CUT&RUN binding buffer (20mM HEPES-KOH pH 7.9, 10mM KCl, 1mM CaCl2, 1mM MnCl2) for 10 min at 4'C. ConA-bead-bound nuclei were incubated overnight at 4'C in CUT&RUN antibody buffer (CUT&RUN wash buffer supplemented with 0.1% triton X-100 and 2mM EDTA) and antibody. After antibody incubation, ConA-bead-bound nuclei were washed once with CUT&RUN triton wash buffer (CUT&RUN wash buffer supplemented with 0.1% triton X-100) then resuspended and incubated at 4'C for 1hr in CUT&RUN antibody buffer and 2.5uL pAG-MNase (EpiCypher). ConA-bound-nuclei were then washed twice with CUT&RUN triton wash buffer and resuspended in 100uL of lysis buffer (50mM Tris-HCl, 150mM NaCl, 1% triton X-100, 0.1% sodium deoxycholate, 5mM CaCl2) and incubated on ice for 30 min. The reaction was stopped by addition of 100ul 2x stop buffer (30mM EGTA) and incubated at 37'C for 10 min. After incubation, ConA-boun-nuclei were captured using a magnet and supernatant containing CUT&RUN DNA fragments was collected. Supernatant was incubated at 70'C for 2hrs with 2uL 10% SDS and 2.5uL 20mg/mL proteinase K. DNA was purified using PCI and overnight ethanol precipitation with glycogen at -20'C. DNA was resuspended in 15uL of buffer EB. CUT&RUN sequencing libraries were generated using NEBNext Ultra II DNA Library Prep Kit for Illumina and NEBNext Multiplex Oligos for Illumina (Index Primer Set 1 and 2). PCR-amplified libraries were purified using 1.2x ratio of AMPure XP beads and eluted in 15uL 0.1x TE buffer NEBNext Ultra II DNA Library Prep Kit for Illumina