RNA-seq Analysis of HNRNPL Knockdown

Control or HNRNPLi cells were harvested 4 days after siRNA transfection. Independent biological duplicates were obtained for both CTLi and HNRNPLi, and total RNA was isolated using the GeneJET RNA purification kit (Thermo Fisher Scientific: K0732) and quantified by Nanodrop. RNA-seq was performed using the Illumina (San Diego, CA) NovaSeq 6000. RNA-seq libraries were prepared with NEBNext Ultra II Library Prep Kit (Illumina (San Diego, CA): E7760) then multiplexed, and approximately 40 million reads per sample were obtained. Pair-end reads were aligned to the GENCODE v19 transcriptome hg19 using TopHat2 with default settings [55 (link)]. Differential expression among samples was calculated using ANOVA from the Partek Genomic Suite (Partek, St. Louis, MO). Analysis of the read count distribution indicated that a threshold of 10 reads per gene generally separated expressed from unexpressed genes, so all genes with fewer than 10 reads were excluded from ANOVA analysis. Gene lists for significantly up-regulated or down-regulated genes were created using p-value < 0.05 and ≥2-fold change. Enriched GO terms for RNA-seq differentially expressed gene sets were identified using Enrichr [56 (link),57 (link)]. Heatmaps for the RNA-seq data were generated using Partek Genomic Suite (http://www.partek.com/partek-genomics-suite/).