title: GSM2825146: nRNA_24_NO; Homo sapiens; RNA-Seq
library_construction_protocol: For ChIP-seq, lysates were clarified from sonicated nuclei and chromatin was isolated and precipitated with antibodies as follows: anti-RNAPII CTD (ser2P) (ab5095, lot GR231750-2) from abcam; anti-RNAPII CTD (ser5P) (3E8; 04-1572-I, lot: 2395817) and anti RNAPII CTD (hypo) (8WG16; 05-952, lot: 2262610) from Millipore. totalRNA was harvested using Trizol reagent. Nacent RNA (nRNA) was obtained according to manufacturer's protocol (Nascent RNA purification kit, Invitrogen) using 5 to 10 minutes for incubation with 100uM EU, mRNA was isolated with polydT dynabeads. Genome-wide capture of damaged DNA (CPD lesion) was achieved by adatation of a MeDIP-seq protocol: single stranded DNA was obtained from reverse crosslinked and purified genomic DNA isolated immediately after UV-C irradiation, then anti-CPD antibody (Anti Cyclobutane Pyrimidine Dimers, clone TDM-2, cat: NMDND001, lot TMC-05) was used to pull down damage DNA before purification and library preparation Libraries were prepared according to Illumina's instructions. ChIP-Seq, nascent RNA-seq ( nRNA-seq) and mRNA-seq ) were prepared by generating fragmented double-stranded cDNA intermediates. In all cases, DNA libraries were generated by ligation adapters and amplification by LM-PCR (Truseq) or (NEBNext). Briefly, double stranded DNA in the range of 100-400 bp was end-repaired using a combination of T4 DNA polymerase, E. coli DNA Pol I large fragment (Klenow polymerase) and T4 polynucleotide kinase. The blunt, phosphorylated ends were treated with Klenow fragment  and dATP to yield a protruding 3- 'A' base for ligation of Illumina's adapters which have a single 'T' base overhang at the 3' end. ALternatively, we used NEBnext and Truseq approaches to multiplex samples for sequencing different samples in a single lane of the flowcell. qPCR with Illumina primers was used to control for overamplification. The purified DNA library was size selected by AMpure beads purification as previously described and was tyoically in the range of 200 to 500 bp. Libraries were sequenced on Hiseq2000 or 2500 in Genecore, EMBL following manufacturer's protocols.
attributes.tag: GEO Accession
attributes.value: GSM2825146