title: GSM1294150: c-Jun knockdown in PC3 cells using shRNa replicate 3; Homo sapiens; RNA-Seq
library_construction_protocol: Total RNA was isolated from PC3 cells transduced with lentiviral shRNA knockdown vectors using the Rneasy mini kit (Qiagen) according to manufacturer's instruction Sequencing libraries were generated using a modified Illumina Truseq sample preparation protocol.Total RNA was DNAase treated with TURBO DNase (Invitrogen) The DNase treated RNA was polyA selected with oligo(dT) beads (Invitrogen). A Superscript III Reverse Transcriptase First-Strand Synthesis (Invitrogen) system was used to generate cDNA from the polyA selected RNA with random hexamer primers (Invitrogen). After first strand synthesis a second strand was generated using E. coli DNA ligase (New England BioLabs) and E. coli DNA polymerase I (New England BioLabs). The double stranded cDNAs were sheared to ~150 nucleotides using a Diagenode BioRuptor and the size was confirmed by DNA gel electrophoresis. DNA end repair of the cDNA was performed using Klenow DNA polymerase (New England BioLabs), T4 DNA polymerase (New England BioLabs), and T4 DNA ligase (New England BioLabs) before the sample was subjected to QIAquick PCR purification (Qiagen). Adapters were ligated to DNA fragments using the T4 DNA ligase (New England BioLabs). The product was run on a 2% agarose gel and size selected between 200 and 300 nucleotides to then be purified by a Gel Extraction kit (Qiagen). Universal and indexing adapters were taken from the TruSeq sample preparation kit (Illumina).
attributes.tag: GEO Accession
attributes.value: GSM1294150