GSM4625018: ChIP-seq-Clone21-TSS-CRISPR-H3K27Ac; Homo sapiens; ChIP-Seq
Chromatin immunoprecipitation (ChIP) was performed as previously described (Hsiung, et al., Genes Dev., 2016), using the H3K27Ac antibody (Active Motif 39685), CTCF antibody (Millipore 07-729), and Rad21 antibody (abcam ab992). Briefly, ~20 million HAP1 cells were fixed in 1% formaldehyde in fresh culture medium at room temperature for 10 minutes, followed by quenching in 1M glycine for 5 minutes. Crosslinked cells were lysed for 10 minutes in 1mL cold Cell Lysis Buffer (10mM Tris pH 8.0, 10mM NaCl, and 0.2% NP-40/Igepal), supplied with Protease Inhibitors (Sigma-Aldrich P8340) and phenylmethylsulfonyl fluoride (PMSF). Nuclei were pelleted, resuspended in 1mL room temperature Nuclei Lysis Buffer (50mM Tris pH 8, 10mM EDTA, 1% SDS), with Protease Inhibitors and PMSF, and were incubated on ice for 20 minutes. The samples were sonicated at 100% amplitude, 30 seconds on/30 seconds off, for 45 minutes, in a bath sonicator (QSonica Q800R3). Sonicated materials were centrifuged, with the supernatant subsequently collected, and diluted with 4mL IP Dilution Buffer (20mM Tris pH 8, 2mM EDTA, 150mM NaCl, 1% Triton X-100, 0.01% SDS), with Protease Inhibitors and PMSF. 50µL protein A/G agarose beads (Thermo Fisher 15918014, Thermo Fisher 15920010) and 50µg isotype-matched IgG control were added to sonicated chromatin to pre-clear it for >2 hours at 4°C. Beads were then spun down, with 200µL supernatant containing pre-cleared chromatin saved as “input” before immunoprecipitation. The remaining pre-cleared chromatin was split into equal volumes, each incubated with antibody or isotype-matched control (IgG) pre-bound protein A/G beads, and rotated overnight at 4°C. Chromatin-bound beads were washed on ice, once with IP Wash 1 (20mM Tris pH 8, 2mM EDTA, 50mM NaCl, 1% Triton X-100, 0.1% SDS), twice with High Salt Buffer (20mM Tris pH 8, 2mM EDTA, 500mM NaCl, 1% Triton X-100, 0.01% SDS), once with IP Wash 2 (10mM Tris pH 8, 1mM EDTA, 0.25M LiCl, 1% NP-40/Igepal, 1% sodium deoxycholate), and twice with TE. Beads were then moved to room temperature, and were eluted twice with a total volume of freshly prepared 200µL Elution Buffer (100mM NaHCO3, 1% SDS). Into each IP and input, 12µL 5M NaCl and 2µL RNase A (10mg/mL, Roche through Sigma 10109169001) were added, and samples were incubated at 65°C overnight. 3µL proteinase K (20mg/mL, Roche through Sigma 3115879) was then added, for an additional 2 hours at 65°C. DNA was column cleaned up using a QIAquick PCR Purification Kit (QIAGEN 28106). For ChIP-sequencing, library construction was performed using Illumina's TruSeq ChIP sample preparation kit (Illumina IP-202-1012), followed by size selection using SPRIselect beads (Beckman Coulter, B23318). Libraries were quality checked, quantified prior to 1 x 75 bp sequencing on the Illumina NextSeq 500.