GSM5379800: ChIP_input_ERHoxB8_CRISPR_region3; Mus musculus; ChIP-Seq
Culture media was removed, and plates were washed once with PBS and then fixed for 10 minutes with 1% formaldehyde (Thermo Fisher Scientific) in PBS at room temperature. Reaction was then quenched by adding glycine (Thermo Fisher Scientific) to 0.125M. After fixation, cells were washed once with cold PBS and then scraped into supernatant using a rubber policeman, pelleted for 5 minutes at 400xg at 4°C. Cells were transferred to Eppendorf DNA LoBind tubes and pelleted at 700xg for 5 minutes at 4°C, snap-frozen in liquid nitrogen and stored at -80°C until ready for ChIP-seq protocol preparation. Chromatin immunoprecipitation (ChIP) was performed in biological replicates as described previously (Hoeksema et al., 2020). Samples were sonicated using a probe sonicator in 500 µl lysis buffer (10 mM Tris/HCl pH 7.5, 100 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 0.1% deoxycholate, 0.5% sarkozyl, 1x protease inhibitor cocktail). After sonication, 10% Triton X-100 was added to 1% final concentration and lysates were spun at full speed for 10 minutes. 1% was taken as input DNA, and immunoprecipitation was carried out overnight with 20 µl Protein A Dynabeads (Invitrogen) and 2 µg specific antibodies for C/EBPβ (Santa Cruz, sc-150), PU.1 (Cell Signaling, #2258), and H3K27ac (Active Motif, 39135). Beads were washed three times each with wash buffer I (20 mM Tris/HCl, 150 mM NaCl, 0.1% SDS, 1% Triton X-100, 2 mM EDTA), wash buffer II (10 mM Tris/HCl, 250 mM LiCl, 1% IGEPAL CA-630, 0.7% Na-deoxycholate, 1 mM EDTA), TE 0.2% Triton X-100 and TE 50 mM NaCl and subsequently resuspended 25 µl 10 mM Tris/HCl pH 8.0 and 0.05% Tween-20. ChIP-seq libraries were prepared on the Dynabeads as described below. For locus specific enrichment ChIP-seq, bead complex was resuspended in 50 µl 1% SDS-TE. 4 µl ProtK, 4 µl RNase A, 3 µl 5 M NaCl was added to these and the input samples and incubated at 50℃ for 1 hour, reverse crosslinked at 65℃ overnight and then eluted from the beads. For wild-type cells, ChIP libraries were prepared while bound to Dynabeads using NEBNext Ultra II Library preparation kit (NEB) using half reactions. DNA was polished, polyA-tailed and ligated after which dual UDI (IDT) or single (Bioo Scientific) barcodes were ligated to it. Libraries were eluted and crosslinks reversed by adding to the 46.5 µl NEB reaction 16 µl water, 4 µl 10% SDS, 4.5 µl 5 M NaCl, 3 µl 0.5 M EDTA, 4 µl 0.2 M EGTA, 1 µl RNAse (10 mg/ml) and 1 µl 20 mg/ml proteinase K, followed by incubation at 55℃ for 1 hour and 75℃ for 30 minutes in a thermal cycler. Dynabeads were removed from the library using a magnet and libraries were cleaned up by adding 2 µl SpeedBeads 3 EDAC (Thermo) in 124 µl 20% PEG 8000/1.5 M NaCl, mixing well, then incubating at room temperature for 10 minutes. SpeedBeads were collected on a magnet and washed two times with 150 µl 80% ethanol for 30 seconds. Beads were collected and ethanol removed following each wash. After the second ethanol wash, beads were air dried and DNA eluted in 12.25 µl 10 mM Tris/HCl pH 8.0 and 0.05% Tween-20. DNA was amplified by PCR for 14 cycles in a 25 µl reaction volume using NEBNext Ultra II PCR master mix and 0.5 µM each Solexa 1GA and Solexa 1GB primers. Libraries were size selected using TBE gels for 200 – 500 bp and DNA eluted using gel diffusion buffer (500 mM ammonium acetate, pH 8.0, 0.1% SDS, 1 mM EDTA, 10 mM magnesium acetate) and purified using ChIP DNA Clean & Concentrator (Zymo Research).    For CRISPR/Cas9-mutated cells, after performing the target-specific ChIPs, we performed an initial PCR for locus-specific amplicon enrichment using NEBNext 2X High Fidelity PCR MM (NEB) and 5'-biotinylated stub adapter primers specific to appropriate genomic regions to be interrogated (Figure 5-table supplement 1). Initial hotstart/denaturation at 98℃ for 30 sec was followed by 10 cycles of amplification (98℃ for 15 sec, 65-67℃ for 15 sec, 72oC for 30 sec) and then a final elongation at 72℃ for 5 min. After this, we performed a 0.7X AmpureXP clean-up and eluted in 20 µl 0.5x TT (5 mM Tris pH 8.0 + 0.025% Tween20). Dynabeads MyOne Streptavidin T1 beads were then washed in 1x Wash Binding Buffer (WBB, 2X WBB: 10 mM Tris-HCl (pH 7.5), 1 mM EDTA, 2 M NaCl, 0.1% Tween) and resuspended beads at 20 µl per sample in 2x WBB. 20 µl prepared Dynabeads MyOne Streptavidin T1 beads (in 2x WBB) were then added to cleaned up 20 µl 0.5x TT PCR fragments, mixed and incubated for 60min at RT with mild shaking. After this, beads were collected on a magnet and washed twice with 150 µl 1x WBB and once with 180 µl TET (TE + 0.05% Tween-20). Finally, beads were resuspended in 25 µl 0.5x TT and on bead PCR for addition of Illumina-specific adapters and 10-bp Unique Dual Indexes (UDIs) using NEBNext 2X High Fidelity PCR MM (NEB) and 25 PCR cycles was performed (Figure 5-table supplement 2). Libraries were size selected using TBE gels for 300-500 bp and DNA eluted using gel diffusion buffer and purified using ChIP DNA Clean & Concentrator (Zymo Research).