RNA-Seq

RNA was extracted using the RNAeasy Mini Kit as per the manufacturer’s protocol (Qiagen). Sample quality was assessed using NanoDrop™ 2000 Spectrophotometers. RNA sequencing was done on Hiseq 4000, PE100bp sequencing platform (BGI, China) using Truseq RNA library (30Million clean reads), and quality was evaluated with the FastQC [46]. To analyze differentially expressed genes after G9a knockdown, the datasets were aligned using STAR (version 2.5.3a) followed by Rsubread (version 1.34.1) and DESeq 2 (version 1.18.1). Data were analyzed based on shG9a independently as well as cohesively, compared to shNT. Genes are considered differentially expressed when the log2foldchange is smaller than -1 and larger than 1, and the p-values are less than 0.05. In pre-ranked gene set enrichment analysis (GSEA), results from DESeq 2 were first ranked based on an algorithm featured by Mark Ziemann (http://genomespot.blogspot.com/2014/09/data-analysis-step-8-pathway-analysis.html). All ‘NA’ results for log2foldchange and p-values were set to 0 and 1 respectively prior to ranking. Non-canonical NF-κB genesets were downloaded from MSigDB [47] with the keywords “NIK OR nfkb2 OR relb”. Ranked genes were then evaluated in the “Pre-ranked GSEA” module featured in GenePattern [48]. The human genome (GRCh37.p13 Release 19) sequences and annotations from Gencode [49] were used as reference.