GSM6787995: EdUseq_ChrRNA; Homo sapiens; OTHER
HeLa S3 cells were fixed in 1% formaldehyde during 10 min and reaction stopped adding glycine (0.125 M) for 5 min at room temperature (RT). Fixed cells were lysed for 20 min at RT in ice-cold lysis buffer (100mM NaCl, 66mM Tris HCl pH8, 5mM EDTA, 0.3% SDS, 1.6% Triton X-100) supplemented with protease inhibitors; passed through a 21G needle and sonicated using a Bioruptor UCD 200 TM-EX (Diagenode) to obtain chromatin fragment size of 250-500 bp. Sonicated chromatin was centrifuged at 19,000 x g at 4°C for 10 min and the supernatant was isolated for subsequent steps. DNA was de-crosslinked and purified with QIAquick PCR Purification kit (QIAGEN). 100 ng of purified EdU labeled DNA were used to perform libraries with KAPA Hyperprep kit following manufacturer's instructions, except that 15 μM xGen Methyl UDI-UMI Adapters (IDT) were ligated during 4 h. Samples were biotinylated performing Click IT reaction during 45 min at RT. Then, MyOne Streptavidin T1 beads (Thermo Fisher Scientific) were used to capture biotinylated libraries and subjected to PCR amplification following KAPA Hyperprep kit protocol (11 PCR cycles). Library construction is performed during the chromatin extraction