RNA-seq Analysis of SIRT3 Knockdown in DLBCL

Total RNA was extracted from DLBCL cell lines expressing control or SIRT3 shRNAs at day 8 after infection using TRIzol (Life Technologies) and RNeasy isolation Kit (Qiagen). RNA concentration was determined using Qubit (Life Technologies), and integrity was verified using Agilent 2100 Bioanalyzer (Agilent Technologies). Libraries were generated using the TruSeq RNA sample kit (Illumina). First-strand synthesis was performed using random oligos and SuperscriptIII (Invitrogen). After second-strand synthesis, a 200-bp paired-end library was prepared following the Illumina paired-end library preparation protocol. Pair-end sequencing (PE50) was performed on Illumina HiSeq2000. RNA-seq results were aligned to hg19 using STAR (47 (link)) and annotated to RefSeq using the Rsubread package (48 (link)). Unsupervised hierarchical clustering was performed using Euclidean distance and Ward's minimum variance method for the top variable genes within each cell line (95th percentile according to SD). Differential expression was determined using edgeR package glmFit function, correcting for cell line using an additive design model (49, 50 (link)). Pathway enrichment of differential expression signatures was calculated using hypergeometric test.