title: GSM7771825: Sample B3; Homo sapiens; RNA-Seq
library_construction_protocol: We used the AllPrep DNA/RNA Mini Kit from Qiagen (#80284) to extract DNA and RNA from each sample. DNA was measured using Qubit fluorometer. RNA quality and quantity were measured using an Agilent 2100 Bioanalyzer. mRNA libraries were generated from 10 ng of RNA from each sample using the TruSeq® RNA Library Prep for Enrichment (20020189) and TruSeq® RNA Enrichment (20020490) library prep kits, following the manufacturer's protocol. The libraries were sequenced on an Illumina NovaSeq 6000 System high-output flow cell (2 × 150bp), with one lane of an S4 flow cell used for the transcriptome sequencing of all 23 biopsies. In total 2,568,575,326 reads were obtained and aligned to the human reference genome (GRCh38) using STAR. Duplicate reads were removed and the remaining reads were sorted using SamTools. Gene expression counts were generated for each sample using featureCounts in the Rsubread package. The resulting counts were normalized to counts per million (CPM) and subjected to trimmed mean of M (TMM) normalization using edgeR. During the TMM normalization step, genes with counts below a certain CPM cutoff were filtered out to reduce noise. On an average, approximately 75% of the reads were uniquely mapped.