GSM8634652: HeLa cells, Nuc RNA-seq, 12A, rep1; Homo sapiens; RNA-Seq
For RNA-seq, Total RNA and RNA associated with different cell fractions were extracted using TRIzol™ reagent (Invitrogen, 15596026) according to the manufacturer's protocol and quantified using NanoDrop One spectrophotometer (Invitrogen). For ChIP-seq, HeLa cells were resuspended with growth medium into a density of 106 mL-1. Crosslinking was performed by adding 1% formaldehyde (Sigma-Aldrich, F1635) into the cells followed by rotation for 20 min at RT, which was ceased by glycine with a final concentration of 0.125 M followed by rotating for 5 min at RT. Supernatants were removed and cell pellets were rinsed with ice cold PBS twice, then the cells were resuspended with lysis buffer (50 mM HEPES, pH 7.9, 5 mM MgCl2, 0.2% Triton X-100, 20% glycerol, 300 mM NaCl, 1× protease inhibitor) on ice for 10 min. Cell pellets were collected by centrifugation at 4 °C, 500 g for 5 min, then resuspended with SDS lysis buffer (50 mM HEPES, pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% sodium deoxycholate, 0.1% SDS, 1× protease inhibitor) and kept on ice for 30 min. The lysates were sonicated utilizing a Bioruptor sonicator (Diagenode) at high power for 30 cycles, followed by centrifugation at 17000 × g for 10 min. Supernatants were collected into new tubes. The specified antibodies, BRD4 (E2A7X) Rabbit mAb (Cell Signaling Technology, 13440S) and HA-Tag (C29F4) Rabbit mAb (Cell Signaling Technology, 3724S), were diluted 1:50 in SDS lysis buffer and incubated with a pre-washed mixture of Protein A and G magnetic beads at 4 °C with continuous rotation for a minimum of 2 h. The beads were subsequently washed three times with SDS lysis buffer. Cell lysates were pre-cleaned with washed Protein A and G magnet beads mixture at 4 °C for 1 h with rotation, and 10% of the lysate were kept as the input control. Then, the remaining lysates and antibody-coated beads were incubated at 4 °C overnight with rotation. The beads were washed with rotation sequentially at 4 °C with the following buffers, 5 min for each: twice with SDS lysis buffer, twice with high salt wash buffer (50 mM HEPES, pH 7.5, 350 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% sodium deoxycholate, 0.1% SDS), twice with LiCl wash buffer (10 mM Tris-HCl, pH 8.0, 250 mM LiCl, 1 mM EDTA, 0.5% NP-40, 0.5% sodium deoxycholate) and once with TE wash buffer (10 mM Tris-HCl, pH 8.0, 1 mM EDTA, 0.2% Triton X-100), then eluted with 240 µL elution buffer (100 mM NaHCO3, 1% freshly added SDS) by shaking at 30 °C for 1 h. The eluent was then mixed with 14.4 µL 5 M NaCl and shaken at 65 °C for 4 h to reverse the crosslinks between proteins and DNA. Afterwards, the eluent was treated with 4 µL RNase A (Thermo Scientific, EN0531), shaken at 37 °C for 15 min, then 4 µL proteinase K (Thermo Scientific, 100005393), shaken at 65 °C overnight. The DNA was extracted by DNA clean & concentrator (Zymo Research, D4014) and set aside for subsequent qPCR assay or library preparation. For RNA-seq, RNA-seq library preparation was performed utilizing VAHTS Universal V8 RNA-seq Library Prep Kit (Vazyme, NR605) following the manufacturer's protocols with 1 µg initial amount of total RNA for each sample, and sequencing was performed at JMDNA Bio-Medical Technology Corporation on an Illumina NovaSeq 6000 platform. For ChIP-seq, library preparation was performed utilizing VAHTS Universal Pro DNA Library Prep Kit (Vazyme, ND608) following the manufacturer's protocols with 10 ng initial amount of DNA for each sample, and sequencing was performed at JMDNA Bio-Medical Technology Corporation on an Illumina NovaSeq 6000 platform.