GSM8625250: ChIP_RNAPII_ETOP; Homo sapiens; ChIP-Seq
RPE-1 cells were crosslinked with 1% formaldehyde for 10 minutes at 37ºC. In METTL3 ChIPseq, double-crosslinking was performed by also adding DSS (disuccinimidyl suberate) in a final concentration of 5 µM in PBS for 5 minutes at 37ºC before formaldehyde addition. Crosslinking reaction was quenched with 0.136 M glycine for 5 minutes at RT. Chromatin was sonicated using Bioruptor Pico (Diagenode) to obtain chromatin fragment size of 250-500 bp. Chromatin associated to each RNAPII was obtained by incubating sonicated chromatin with the corresponding antibody. DNA was purified using QIAquick® PCR Purification Kit (QIAGEN). Two independient replicates were performed for RNAPII ChIPseq and only one for METTL3 ChIPseq. ChIPmentation was carried out using Tn5A Enzyme during the  and libraries were amplified using NEBNext® High-Fidelity 2X PCR Master Mix (NEB). Libraries were purified with Sera-Mag Select Beads (CYTIVA).