Probing the Global Cellular Responses to Lipotoxicity Caused by Saturated Fatty Acids

RNA sequencing technology was applied to determine the transcriptional changes due to palmitate treatment, RNF213 knockdown, and GPAT4 knockdown. Total RNA was isolated from K562 cells using an RNeasy Kit. The library construction and sequencing were performed by the TUFTS sequencing core (tucf-genomics.tufts.edu/). Briefly, the RNA was run on a Bioanlayzer (Agilent) to assess RNA quality and quantification. The biological triplicates were pooled at this point. The library preparation was performed using the TruSeq Stranded mRNA Library Prep Kit (Illumina). The samples were sequenced by HiSeq 2500, Rapid Paired-End 100 (RL-PE100) or HiSeq 2500, High Output v4 Paired-End 100 (HI-PE100, Illumina). All reads were mapped to the UCSC hg19 human reference genome using Tophat2 with multi-read correction, followed by normalization, quantification and differential expression analysis using Cuffdiff.