GSM5668638: ChIP-seq_C65_Smc1a; Mus musculus; ChIP-Seq
Cells were cross-linked for 5 minutes at room temperature with 1% formaldehyde-containing medium; cross-linking was stopped by PBS-glycine (0.125 M final). Cells were washed twice with ice-cold PBS, scraped, centrifuged for 10 min at 4000 rpm, resuspended in cell lysis buffer (5 mM PIPES, pH 8.0, 85 mM KCl, and 0.5% NP-40, 1 ml/15 cm plate) and incubated for 10 min on ice. During the incubation, the lysates were repeatedly pipetted up and down every 5 minutes. Lysates were then centrifuged for 10 min at 4000 rpm. Nuclear pellets were resuspended in 6 volumes of sonication buffer (50 mM Tris-HCl, pH 8.1, 10 mM EDTA, 0.1% SDS), incubated on ice for 10 min, and sonicated to obtain DNA fragments below 2000 bp in length (Covaris S220 sonicator, 20% Duty factor, 200 cycles/burst, 150 peak incident power, 40 cycles of 20" on and 40" off). Sonicated lysates were cleared by centrifugation and 800 g of chromatin were diluted in RIPA buffer (10 mM Tris-HCl, pH 8.0, 1 mM EDTA, 0.5 mM EGTA, 1% Triton X-100, 0.1% SDS, 0.1% Na-deoxycholate, 140 mM NaCl) to a final concentration of 0.8 ug/ul, precleared with Protein A sepharose (GE Healthcare) for 2 hours at 4°C and immunoprecipitated overnight with 8 ug of normal rabbit IgGs (ChromPure rabbit normal IgG; Jackson ImmunoResearch), anti-Smc1a (A300-055A; Bethyl Laboratories Inc.), or 1.6 ug of anti-CTCF antibodies (ab128873; Abcam). About 8% of the precleared chromatin was saved as input. Immunoprecipitated DNA was purified with the Qiagen QIAquick PCR Purification Kit and eluted in 60 ul of water. ChIP-seq libraries were prepared using the Solexa rapid library protocol. Immunoprecipitated DNA or 50 ng input DNA was end-repaired, phosphorylated and adenylated in a single 50 μl reaction containing 31.5 μl DNA, 5 μl spike-in yeast DNA from MNase treated nucleosomes (10 ng/ml) and 13.5 μl end-repair/3' A mix (1X T4 DNA ligase buffer, 0.5 mM dNTPs, 0.25 mM ATP  2.5% PEG 4000, 0.0025 U/μl T4 PNK, 0.0025 U/μL T4 DNA polymerase, 0.0025 U/μL Taq DNA polymerase). The reaction was incubated in a thermal cycler for 15' at 12'C, 15' at 37'C, 20' at 72'C, and held at 4'C. Reactions were added of 4 μl water, 1 μl Illumina TruSeq adapters, 55 μl 2x Rapid DNA ligase buffer (Enzymatics #B101L) and 5 µl DNA ligase (Enzymatics #L6030-HC-L), and incubated for 15' at 20'C. Ligations were cleaned up twice with AMPure XP beads (Agencourt #A63880) diluted 1:2 with 20% PEG, 1.25M NaCl (first cleanup: 38 μl; beads eluted with 53 μl 10 mM Tris-HCl pH 8.0, 50 μl transferred to a new tube and added of 55 μl beads:PEG solution). Final elution volume was in 22 μl 10 mM Tris-HCl pH 8.0, 20 μl of which were transferred to a new tube and amplified by PCR (45" at 98'C; 14 cycles of 15" at 98'C and 10" at 60'C; 1' at 72'C; hold at 4'C) with KAPA HS HIFI polymerase (1X KAPA buffer, 0.3 mM dNTPs, 0.5 μM TruSeq PCR primers, 1U KAPA polymerase). PCR reactions were cleaned up once with 38 μl AMPure XP beads diluted 1:2 with 20% PEG, 1.25M NaCl and eluted with 33 μl 10 mM Tris-HCl pH 8.0, 30 μl of which were transferred to a new tube.