GSM6787998: EdUseq_HU_ChORseq; Homo sapiens; OTHER
HeLa S3 cells from each labeling set-up 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 and phosphatase 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. In parallel, Drosophila S2 cells were fixed, lysed and sonicated as described above. After sonication, Drosophila S2 sonicated chromatin was added to HeLa S3 chromatin in a proportion of 0.05% of total HeLa S3 chromatin as internal control. Chromatin associated to S2P RNAPII was obtained by incubating sonicated chromatin with the corresponding antibody. DNA from ChIPs was purified using MiniElute PCR purification kit (QIAGEN) where the five ChIPs from each condition were purified together in the first step. 17 ng of immunoprecipitated DNA for each time point sample were used to perform libraries with KAPA Hyperprep kit following manufacturer's instructions, except that 7.5 μM xGen Methyl UDI-UMI Adapters (IDT) were ligated during 4 h. Later, indexed samples from both biological replicates (T0 rep1, T1 rep1, T0 rep2, T1 rep2) were pooled together and 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 (14 PCR cycles were used). For EdU-seq, HeLa S3 and Drosophila S2 mixed chromatin (0.05% of total volume) from T0 was de-crosslinked and purified with QIAquick PCR Purification kit (QIAGEN). Libraries and streptavidin pull downs of EdU-seq experiments were performed as described above but using 100 ng of purified EdU labeled DNA, 15 μM of xGen Methyl UDI-UMI Adapters and performing 11 cycles of PCR amplification. Library construction is performed during the chromatin extraction