Paired end library preparation protocol

Paired-end (PE) libraries were prepared according to the Illumina paired-end library preparation protocol (Illumina, San Diego, CA), and were sequenced on an Illumina Hiseq. 2000 sequencing system to generate 2 × 90 PE reads. High-quality reads of PE were obtained for each sample. Adaptor sequences, reads with more than 10% unknown sequences, “N”, and low-quality sequences (the percentage of low-quality bases with a threshold quality score <20) were removed. The obtained sequence reads were quality-checked by FastQC⁴⁴ . Clean reads were aligned to the porcine reference genome sequence (Sscrofa11.1) using TopHat v2.0.1 software^{45 (link)}. The porcine genome (Sscrofa11.1) was obtained from Ensembl database. Reads aligned to the reference genome were assembled by Cufflinks software^{46 (link)}. Cuffdiff, a part of the Cufflinks package, was used to identify differentially expressed genes (DEGs) and transcripts from two groups of pigs with opposing backfat thickness performance^{47 (link)}. The threshold value for selection of differentially expressed genes is q-value ≤ 0.05 and fold change (FC) ≥ 2 or ≤ 0.5, as |Log₂ FC| ≥ 1. Moreover, DEGs between pigs in each pair were also analyzed using the same analysis pipeline.

Total RNA was extract from patient samples using Trizol (LifeTechnologies). RNA concentration was determined using Qubit (LifeTechnologies) and integrity was verified using Agilent 2100 Bioanalyzer (Agilent Technologies). Libraries were generated using mRNA-seq sample prep kit (Illumina), through which mRNA was selected by two rounds of purification using magnetic polydT beads and then fragmented. 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.

Total RNA was extracted using Trizol reagent (Invitrogen, USA), in accordance with the manufacturer’s instructions. RNA quality was assessed by electrophoresis on a 1% agarose gel and with an Agilent 2100 Bioanalyzer (Agilent, USA). mRNA was isolated from total RNA using oligo(dT) magnetic beads and disrupted into short fragments of about 300 bp. Paired-end (PE) libraries were prepared according to the Illumina paired-end library preparation protocol (Illumina, USA). PE libraries were sequenced on an Illumina Hiseq 2000 sequencing system to generate 2 × 125 PE reads at Beijing Genomics Institute at Shenzhen.

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.