GSM5768810: LKO-PAF; Homo sapiens; RNA-Seq
For next generation sequencing RNA-seq analysis, total RNA was extracted from normal fibroblasts (NFs), para-cancer-associated fibroblasts (PAFs), and cancer-associated fibroblasts (CAFs) using TRIzol reagent (Invitrogen, Carlsbad, CA, USA). RNA quality control was performed with the Agilent Bioanalyzer system (Agilent Technologies, Santa Clara, CA, USA). The sequencing library was prepared with Truseq Stranded Total RNA Sample Preparation Kit (Illumina, San Diego, CA, USA), followed with library quality check using Agilent Bioanalyzer system. Sequencing was performed on Illumina HiSeq2000 machines (Illumina) using the standard Illumina RNAseq protocol with a read length of 2 x 100 bases. All sequenced reads were quality checked using FastQC followed by mapping to the human reference genome (Genome Research Consortium Human Build 38 [GRCh38], hg38) and the Ensembl release-102 version 73 gene annotation using HISAT2 (Hierarchical Indexing for Spliced Alignment of Transcripts 2) (v2.2.1). To compare expression between genes within samples, gene expression was estimated using StringTie v2.1.4 (v2.1.4) from the “new Tuxedo” package. Gene abundances were normalized to library and gene length by calculating fragments per kilobase of exon per million mapped reads using featureCounts and differentially expressed genes were identified by count data with DESeq2.