GSM8413582: HeLa, RNAPII, siPNUTS, ChIP, rep1; Homo sapiens; ChIP-Seq
Chromatin immunoprecipitation was performed using the iDeal ChIP-seq kit for Transcription Factors (Diagenode, C01010055) according to the manufacturer's instructions. Briefly, 2.5 million HeLa cells per IP were crosslinked with 1% formaldehyde (Thermo Scientific™, 28908) in DMEM for 10 min at room temperature. Chromatin from lysed nuclei was sheared to 200–300 bp fragments using Bioruptor® Pico sonication device (Diagenode, B01060010) and cleared by centrifugation. Sheared chromatin was incubated overnight at 4°C with 3 µg of antibody coupled to the DiaMag protein A-coated magnetic beads (from Diagenode kit). Beads and immunocomplexes were then washed and eluted per the manufacturer's instructions. For PNUTS ChIP-seq, an additional crosslink with the ChIP cross-link Gold reagent (Diagenode, C01019027) was performed before formaldehyde crosslinking. Sequencing libraries from immunoprecipitated DNA were prepared using the MicroPlex kit v2 (Diagenode) according to the manufacturer's protocol. In brief, immunoprecipitated DNA was quantified using Qubit fluorometer (Thermofisher), and 1.5 to 35 ng (for SPT6, IWS1 and RNAPII ChIP) or 2 to 10 ng (for PNUTS ChIP) were used as input material. DNA was repaired and end-blunted by enzymatic treatment. Stem-loop adaptors with blocked 5' ends were ligated to the 5' end of the genomic DNA, leaving a nick at the 3' end. The 3' ends of the genomic DNA are extended to complete library synthesis and Illumina-compatible indexes were added through amplification. Libraries were purified using AMPure XP beads protocol (Beckman Coulter, Indianapolis, IN) and quantified using Qubit fluorometer. Libraries' fragment size distribution was verified using the Bioanalyzer High Sensitivity DNA chip (Agilent Technologies, Santa Clara, CA). Libraries were pooled and spike-in PhiX Control v3 (Illumina, San Diego, CA) was added to represent 1% of the total PF reads.