RNA-Seq of YUMMER1.7
Approximately 8 x 106 cells per sample were treated with 10 M RSL3 or 100 M simvastatin for 18 hours. Cells were then harvested by trypsinization, washed, and RNA was isolated using RNeasy kit, supplemented with QIAshredder and DNase treatment (Qiagen). RNA concentration and fragment sizes were evaluated for QC using BioAnalyzer or Agilent TapeStation, then library prep performed and sequenced using Ilumina 97 HiSeq4000 by the Yale Stem Cell Center Genomics Core. Reads were aligned using STAR244 (v2.7.9) to mm10. Briefly, counts were normalized to library size using cpm() and differential expression analysis calculated by Exact test statistic using exactTest() in a pairwise-manner. Trimmed mean of M-values (TMM) normalization was performed during differential expression analysis with edgeR (v4.36.0). Gene set enrichment analysis was performed using desktop software (v4.2.3) on pre-ranked lists, calculated by sign (logFC) * -log10(pval); and the HALLMARK gene sets from MSigDB (v2022.1)70,71.