title: GSM7965449: ZNHIT1_WT_dTAG_rep2; Homo sapiens; ChIP-Seq
library_construction_protocol: Cells were disrupted using lysis buffer containing 50 mM HEPES pH 8.0, 300 mM NaCl, 5% (v/v) Glycerol, 0.2% (w/v) chaps, 2 mM MgCl2, 0.5 mM EDTA (Ethylenediaminetetraacetic Acid), 2 mM DTT (Dithiothreitol), 1 mM PMSF (Phenylmethylsulfonyl fluoride), 1 μg/mL Aprotinin, 1 μg/mL Pepstatin, 1 μg/mL Leupeptin for 30 min. After centrifuging at 17,000 x g for 15 min, supernatant was collected to incubate with Flag-M2 beads (Sigma) for 4 hours. After immunoprecipitation, protein was washed for three times with wash buffer mentioned above then eluted using wash buffer containing 400 ng/μl Flag peptide (Meilunbio) additionally. All the steps were performed at 4°C. For each ChIP assay, 1 x 107 DLD-1 cells were used, as described, and 1 x 106 MEF cells were mixed with DLD-1 cells as the spike-in. The mixed supernatant was incubated with 3ug antibody overnight. The libraries were prepared with the VAHTS Universal Plus DNA Library Prep Kit for Illumina (Vazyme). Briefly, DNA was end-repaired using a combination of T4 DNA polymerase, E. coli DNA Pol I large fragment (Klenow polymerase) and T4 polynucleotide kinase. The blunt, phosphorylated ends were treated with Klenow fragment (32 to 52 exo minus) and dATP to yield a protruding 3- 'A' base for ligation of Illumina's adapters which have a single 'T' base overhang at the 3' end. After adapter ligation DNA was PCR amplified with Illumina primers for 15 cycles and library fragments of ~250 bp (insert plus adaptor and PCR primer sequences) were band isolated from an agarose gel. The purified DNA was captured on an Illumina flow cell for cluster generation. Libraries were sequenced on the DNBSEQ-T7.