GSM9360618: D6_Ctrl_ETS1_rep2; Mus musculus; OTHER
500,000 FACS-purified EpdSCs per biological replicate per target antibody, with two biological replicates per condition and 2-3 mice pooled per biological replicate. Cells were lightly fixed 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. Crosslinking was quenched with glycine (0.125M final concentration) and rotated at room temperature for 5 min. Cells were washed with cold PBS and nuclei isolated by resuspending in NE1 buffer (20mM HEPES-KOH pH7.9, 10mM KCl, 1mM MgCl2, 1mM DTT, 0.1% Triton X-100, with Roche complete EDTA-free protease inhibitor) and rotated at 4°C for 10 min. Nuclei were washed twice with wash buffer (20mM HEPES pH7.5, 150mM NaCl, 0.5% BSA, 0.5mM spermidine, with protease inhibitor) then incubated with concanavalin-A (ConA) beads previously washed with 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 antibody buffer (Wash buffer supplemented with 0.1% Triton X-100 and 2nM EDTA) and antibody (c-FOS: Cell Signaling 1:50; c-JUN: Cell Signaling 1:50; deltaNP63: BioLegend 1:10). ConA-bound nuclei were washed once with triton wash buffer (wash buffer supplemented with 0.1% Triton X-100) then resuspended in antibody wash buffer with 2.5uL pAG-MNase (EpiCypher) and incubated at 4°C for 1 hr. ConA-bound nuclei were washed twice with 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 iced metal block (0°C) for 30min. MNase reaction was stopped with addition of 100uL 2x stop buffer (30mM EGTA) and incubated at 37°C for 10min. After incubation, ConA-bound nuclei were captured using a magnetic tube holder and supernatant containing CUT&RUN DNA fragments was collected. Supernatant was incubated with 2uL 10% SDS and 2.5uL 20mg/mL proteinase K at 70°C for 2hrs. DNA was purified using PCI and overnight ethanol precipitation with glycogen at -20°C. DNA was spun down and washed once with 100% ethanol before being resuspended in 15uL of buffer EB. Sequencing libraries were generated using NEBNext Ultra II DNA library Prep Kit for Illumina (Index Primer set 2). PCR-amplified libraries were purified using 1.2x ratio of AMPure XP beads and eluted in 15uL 0.1x TE buffer. Libraries were pooled and sequenced on Illumina NextSeq using 40-bp paired-end reads. NEBNext Ultra II DNA Library Prep Kit for Illumina