GSM4593589: TT-seq siINTS11 rep 2; Homo sapiens; OTHER
TT-seq/RNA-seq: Six in vitro transcribed spike-in RNAs were added to the lysates (TRIzol, Thermo) followed by extraction of RNA according to the manufacturer's protocol. The RNA was then sonicated and used as input for RNA-seq or for further purification of 4sU-labeled RNA. 4sU-labeled RNA was first biotinylated with EZ-Link HPDP-Biotin (Pierce) and isolated with µMACS Streptavidin Kit (Miltenyi). 4sU-labeld RNA was eluted by cleaving off the biotin with DTT. Both total RNA and 4sU-labeled RNA purified prior to libary preparation using the miRNeasy Micro Kit (Qiagen) including DNse I treatment to remove genomic DNA. 3'-seq: TT-seq/RNA-seq RNA libraries were prepared for sequencing using standard Illumina protocols (TruSeq Stranded Total RNA Sample Prep kit). 3'-seq libraries were generated with standard and modified Lexogen 3'-seq protocols. The modified preapration included 3'end polydenylation in vitro using E.coli poly(A) polymerase ('xPAP') preceeding the NGS library preparation with the QuantSeq REV kit according to manufacturer's protocol (Lexogen). RNA-Seq, paired-end; RNA 3'end seq, single-end
GEO
GSM4593589