title: GSM4466724: C7_CAGE_seq_Rep3; Mus musculus; RNA-Seq
library_construction_protocol: For the extraction of nuclear RNA, cells were lysed for 5 minutes on ice in lysis buffer (20 mM Tris pH 7.5, 150 mM NaCl, 5% glycerol, 0.5% NP-40, 1x protease inhibitor) then centrifuged at 2000 rpm to pellet nuclei. The nuclei were washed once in cold PBS then directly lysed in Trizol followed by standard RNA extraction. Libraries for all samples (E14 ´ 3, PWWP2A/B DKO C7 ´ 3, DKO A1 ´ 2) were prepared using the CAGE-seq kit purchased from cage-seq.com. Briefly, 5 mg of nuclear RNA for each sample was primed (random) and reverse transcribed to cDNA, followed by biotinylation of and capture of the 7-methylguanosine cap of all RNA Polymerase II transcripts. Adapters containing barcodes (3-letter) were ligated and samples were multiplexed. As this CAGE-seq protocol involves no PCR amplification step, the entire sample was denatured and loaded into the Illumina NextSeq500 for single end 80bp sequencing.
attributes.tag: GEO Accession
attributes.value: GSM4466724