title: GSM2308767: HCMV infected Non-targeting siRNA treated human foreskin fibroblasts [RNA-Seq]; Homo sapiens; RNA-Seq
library_construction_protocol: Total RNA was isolated using TRIzol reagent using the manufacturer’s protocol (Life Technologies) For RNA-seq, libraries were made using the Illumina polyA selection truseq library kit using different barcodes. Libraries were not strand specific. TAIL-seq was performed as described by Chang et al, (2014, Molecular Cell), with minor modifications. Briefly, total RNA was extracted from cells by TRIzol reagent (Invitrogen, 15596-018) according to the manufacturer’s protocol. The RNA ligated to 3′ “splint” oligonulceotide (NNNGTCAGTTTTTTTTT) to enrich for polyadenylated transcripts and partially digested by RNase T1 (Ambion, AM2283). The fragmented RNAs were pulled down with Dynabeads M-280 Streptavidin (Invitrogen, 11206D) magnetic beads, phosphorylated on bead using T4-PNK reaction (NEB), eluted using 2X RNA loading dye (with 95% formamide), run on a 6% polyacrylamide urea gel (NuPage), stained with SYBR gold (Thermo) and gel purified in the range of 250 - 750 nucleotides. The purified RNAs were ligated to 5′ adapter (Illumina Truseq small RNA kit), reverse transcribed using Superscript III (Invitrogen) and amplified by PCR using Phusion DNA polymerase (Thermo, F-530L) and Illumina Truseq small RNA universal forward and indexed (barcode) reverse primer(s). The library was purified using AMPureXP beads (Agilent) and the libraries were sequenced on an Illumina Miseq instrument (51 ~ 251 paired end run) with 10% of the PhiX control library (Illumina, FC-110-3001) and 1% of the spike-ins mixture of known polyA-containing DNAs Non-strand specific. TAIL-seq using Miseq
attributes.tag: GEO Accession
attributes.value: GSM2308767