GSM9376418: Input_R4KO_2A_Rep1; Homo sapiens; ChIP-Seq
Briefly, 10 million HeLa cells mixed with 1 million MOC1 cells were fixed with 1% formaldehyde for 10 min at room temperature, followed by quenching with 125 mM glycine for 5 min. After two washes with ice-cold PBS, cells were pelleted (2,000 g, 5 min) and lysed in SDS lysis buffer (1% SDS, 5 mM EDTA, 50 mM Tris-HCl, pH 8.1, 1 × protease inhibitor). Chromatin was then sonicated for 30 cycles (30 sec on/30 sec off) using a Bioruptor (Diagenode) in an ice-water bath to yield fragments averaging 300 bp. After centrifugation, the supernatant was diluted with a 10-fold volume of dilution buffer (1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl, pH 8.1, 150 mM NaCl and 1 × protease inhibitors). For immunoprecipitation, the diluted chromatin was incubated with 2 g RPB1 antibodies (Cell Signaling Technology) overnight at 4 ºC. On the following day, 30 l of pre-washed Protein G Dynabeads (Invitrogen) were added to the supernatant, and incubated at 4 ºC for 2 hr with rotation The bead-bound complexes were then washed sequentially with buffer I (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl, pH 8.1, and 150 mM NaCl), buffer II (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl, pH 8.1, and 500 mM NaCl), buffer III (0.25 M LiCl, 1% Nonidet P-40, 1% sodium deoxycholate, 1 mM EDTA, and 10 mM Tris-HCl, pH 8.1), and buffer IV (1 mM EDTA and 10 mM Tris-HCl, pH 8.1). Chromatin was eluted by incubating the beads in elution Buffer (50 mM Tris-HCl, pH 8.0, 10 mM EDTA, 1% SDS) supplemented with Proteinase K at 65 °C overnight to reverse cross-links. The eluted DNA was purified by phenol: chloroform: isoamyl alcohol extraction and dissolved in ddH2O. Input DNA was processed in parallel, undergoing de-crosslinking and purification identical to the ChIP samples. DNA concentrations were measured using the Equalbit 1 × dsDNA HS Assay Kit (Vazyme). For library preparation, approximately 10 ng ChIP DNA and 20 ng Input DNA were used to construct libraries according to the VAHTS Universal DNA Library Prep Kit for Illumina (Vazyme). Finally, sequencing was performed on an Illumina NovaSeq 6000 platform (Novogene) with 150-bp paired-end reads.