GSM8897945: U373_NT-shRNA; Homo sapiens; RNA-Seq
The U87 and U373 glioblastoma cell lines were transduced with either a non-targeting short hairpin RNA (shRNA) (designated as NT) or a YB-1-targeting shRNA (denoted as shYB-1) lentivirus. Upon reaching approximately 90% confluency, the cells were harvested and lysed directly in 10 cm² culture dishes utilizing TRIzol reagent (Beyotime). The resulting cell lysates were promptly frozen at -80℃ and subsequently transported on dry ice to Majorbio Bio-pharm Technology Co., Ltd. (Shanghai, China). RNA purification, reverse transcription, library construction and sequencing were performed by Majorbio according to the manufacturer's instructions (Illumina, San Diego, CA). The  RNA-seq transcriptome librariy was prepared following Illumina® Stranded mRNA Prep, Ligation from Illumina (San Diego, CA) using 1μg of total RNA.  Shortly, messenger RNA was isolated according to polyA selection method by oligo(dT) beads and then fragmented by fragmentation buffer firstly. Secondly double-stranded cDNA was synthesized using a SuperScript double-stranded cDNA synthesis kit (Invitrogen, CA) with random hexamer primers (Illumina). Then the synthesized cDNA was subjected to end-repair, phosphorylation and 'A' base addition according to Illumina's library construction protocol. Libraries were size selected for cDNA target fragments of 300 bp on 2% Low Range Ultra Agarose followed by PCR amplified using Phusion DNA polymerase (NEB) for 15 PCR cycles. After quantified by Qubit 4.0, paired-end RNA-seq sequencing library was sequenced with the NovaSeq 6000 sequencer (2 × 150bp read length).