GSM4625050: ChIP-seq-Clone21-CTCF&TSS-CRISPR#2-Rad21-rep2; Homo sapiens; ChIP-Seq
Sample information curated by ChIP-Atlas
Cell type Class
Leukemia Chronic Myelogenous
Attributes by original data submitter
KBM7-derived near haploid cells
Sequenced DNA Library
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. See "extract protocol"