title: RNA-seq of MDA-MB-231 WT clone 2
design: Total RNA from MDA-MB-231 WT and LINC01605-KO cells were extracted using the RNeasy Plus Mini Kit (QIAGEN) according to protocol instructions. Extraction was followed by DNase digestion (Turbo-Dnase, Ambion, Thermo Fisher Scientific, Waltham, MA, USA) and RNA quality was assessed by using an agarose gel electrophoresis after RNA exposure to 70C for 5 minutes. Truseq Stranded mRNA Sample Prep Kit was used for library preparation. Libraries were sequenced on single-end 75 bp mode on NextSeq 500 (Illumina) by IGA (Udine, Italy). To generate cell lines KO for LINC01605 four different single guide RNAs (sgRNA) were used spanning approximately 800 bp of LINC01605 first exon. Briefly, sgRNAs were cloned into pLV hUbC-Cas9-T2A-GFP. MDA-MB-231 cells tranduced with this lentiviral system were sorted by flow citometry in GFP- (wt) and GFP+ (KO) cells.