Neb library quantification kits

RNA was extracted by using RNeasy kit (Qiagen, Hilden, Germany; catalog no. 74014) following the manufacturer’s instructions. All RNA-seq libraries were prepared by using the NEBNext Poly(A) mRNA Magnetic Isolation Module followed by NEBNext Ultra Directional RNA Library Prep Kit for Illumina (both from New England Biolabs, Ipswitch, MA). Library quality was analyzed by using Agilent BioAnalyzer 2100 (Agilent, Santa Clara, CA), and libraries were quantified by using NEB Library Quantification Kits (New England Biolabs). Libraries were then sequenced by using a NextSeq500 platform (75 base pairs, single-end reads) (Illumina). All RNA-seq was aligned by using RNA STAR under default settings to Homo sapiens UCSC hg19 (RefSeq & Gencode gene annotations). FPKM generation and differential expression analysis were performed by using DESeq2. DESeq2 output was analyzed by comparing differentially expressed genes with P <.05, and HALLMARK gene set enrichment analysis was performed by using MSigDB database.³¹ (link) All RNAseq data are deposited on GEO under accession GSE117312.

RNA was extracted by using an RNeasy kit (Qiagen, Hilden, Germany; catalog no. 74014) following the manufacturer’s instructions. All RNA-seq libraries were prepared by using the NEBNext Poly(A) mRNA Magnetic Isolation Module followed by NEBNext Ultra Directional RNA Library Prep Kit for Illumina (both from New England Biolabs, Ipswich, MA). Library quality was analyzed by using Agilent BioAnalyzer 2100 (Agilent, Santa Clara, CA), and libraries were quantified by using NEB Library Quantification Kits (New England Biolabs). Libraries were then sequenced by using a NextSeq500 platform (75 base pairs, single-end reads) (Illumina). All RNA-seq was aligned by using RNA STAR under default settings to Homo sapiens UCSC hg19 (RefSeq and Gencode gene annotations). FPKM generation and differential expression analysis were performed by using DESeq2.