GSM2572696: Bru-seq_Induced_30min_after_induction; Homo sapiens; OTHER
BLISS: Briefly, after fixation, cells were lysed and submitted to in situ DNA ends blunting (with Quick Blunting kit, NEB), followed by in situ DNA ends ligation with BLISS linker. After washing, genomic DNA was extracted and sonicated with Covaris S220 (10% duty factor, 175W peak incident power, 200 Cycles/burst, 105s) to obtain a pool of 300 bp fragments. Afterwards, fragmented DNA was in vitro transcribed using MessageAmpII kit (Ambion) for 14h at 37°C. After RNA purification and ligation of the 3’ Illumina adapter, the RNA was reverse-transcribed. CAGE: we used the mirVana Kit (Ambion) according to the manufacturer’s instructions for the collection of total RNA. RNA-seq: total RNA was isolated using the mirVana Kit (Ambion), according to the manufacturer’s instructions. RNA-seq of DMSO- and ATMi-treated cells: total RNA was isolated using the RNeasy Kit (QIAGEN), according to the manufacturer’s instructions. Bru-seq: cells were incubated at different times after AsiSI induction in 2 mM bromouridine (Bru) for 30 min and total RNA was isolated using TRIzol (Invitrogen) according to the manufacturer’s instructions. ChIP-seq: Lysates were clarified from sonicated nuclei and protein-DNA complexes were isolated with specific antibody against total RNAPII (Millipore, 05-623) or RNAPII pSer2 (Abcam, 5095). BLISS: The final step of library indexing and amplification was performed using the TruSeq Small RNA Library Prep Kit (Illumina, RS-200-0012). Bru-seq: strand-specific DNA libraries with the Illumina TruSeq Kit (Illumina). ChIP-seq: library preparation and sequencing using standard Illumina TruSeq for HiSeq 2000 reagents. RNA-seq: strand-specific library preparation was performed using ScriptSeq v2 RNA-Seq Library Preparation Kit (Epicentre). rRNAs were removed using Ribo-Zero Magnetic Kit (Epicentre). RNA-seq of DMSO- and ATMi-treated cells: strand-specific library preparation was performed using Illumina TruSeq kit. CAGE: cap analysis of gene expression (CAGE) for high-throughput gene expression profiling was performed following nAnT-iCAGE protocol workflow (Murata et al., 2014).