GSM4083123: siGFP_noPAP_in_batch2; Homo sapiens; RNA-Seq
RNAs were extracted using TRIzol (Invitrogen) according to the manufacturer's protocol and then treated with TURBO DNase (Invitrogen) following the manufacturer's instruction to remove contaminating genomic DNA. Six in vitro transcribed spike-in RNAs were added. Sequences for spike-ins are identical to ERCC spike-ins. ERCC-00092, ERCC-00136 and ERCC-00043 were transcribed in the presence of 4sU, whereas ERCC-00170, ERCC-00002 and ERCC-00145 were transcribed without 4sU and serve as negative control in the 4sU purification step. An aliquot of  each RNA was analyzed as total RNA, spiked with an additional full complement of commercially available ERCC spike-ins. The remainder of RNA was used for 4sU purification. Half of all total and 4sU labelled RNA samples were 3'end polydenylated in vitro using E.coli poly(A) polymerase ('xPAP'). All samples were rRNA depleted and subjected to pA+ RNA 3'end sequencing using QuantSeq REV kit from Lexogen GmbH. 8 or 10 libraries were multiplexed and sequenced at the Vienna Biocenter Core Facilities with a NextSeq SR75 High Output run. RNA 3'end seq, single-end