GSM6430598: HeLa, PBS,Hcar1 C-term flag replicate 1; Homo sapiens; ChIP-Seq
Chromatin immunoprecipitation (ChIP) was performed using SimpleChIP® Enzymatic Chromatin IP Kit (Cell Signaling) based on manufacturer's protocol. Briefly, DNA-protein complexes were crosslinked using 1% final concentration of formaldehyde (Sigma) for 10min at RT, and quenched with Glycine (final concentration of 125mM). Cells were then washed with cold PBS + PI cocktail, scraped into conical tubes and centrifuged to remove the supernatant. After isolation, nuclei were treated with micrococcal nuclease to digest the DNA, and then sonicated. Digested chromatin was analyzed by agarose gel. Chromatins were then incubated with immunoprecipitating antibody O/N at 4°C with rotation. Control samples were incubated with IgG antibody. ChIP-grade protein G magnetic beads were added to the IP reactions and incubated for 2h at 4°C with rotation. Beads were then washed with low- to high-salt wash buffers and chromatin was eluted in elution buffer for 30min at 65°C. Chromatins were reverse-crosslinked with NaCl and proteinase K and incubation at 65°C for 2h. DNA was purified with spin columns, and analyzed by qPCR or sent to Next Generation Sequencing. ChIP-seq. Samples were analyzed with bioanalyzer for quality control and single-end NGS was performed at IRIC genomic platform with Nextseq 500 illumina system. ChIP-seq. Samples were sequenced with a depth of ~35M per sample with 75 cycles. Both C- and N-terminus flag tagged cells were used for ChIP-seq studies. Two samples from each terminus tagged cells were used for either lactate or vehicle (PBS); in total 4 samples per treatment group, and only shared genes in each group was used for further bioinformatic analysis.