Transcriptomic Analysis of IFNγ and TNFα Responses in Ptpn2-null B16 Cells

Ptpn2-null or control sgRNA-transfected B16 cells were stimulated with IFNγ (100ng ml⁻¹, Cell Signaling Technology), TNFα (10 ng ml⁻¹, Peprotech) or both for 48 h. RNA was extracted from cell pellets using the Qiagen RNeasy Mini kit according to the manufacturer’s instructions. First-strand Illumina-barcoded libraries were generated using the NEB RNA Ultra Directional kit according to the manufacturer’s instructions, including a 12-cycle PCR enrichment. Libraries were sequenced on an Illumina NextSeq 500 instrument using paired-end 37 bp reads. Data were trimmed for quality using the Trimmomatic pipeline with the following parameters: LEADING:15 TRAILING:15 SLIDINGWINDOW:4:15 MINLEN:16. Data were aligned to mouse reference genome mm10 using Bowtie2. HTSeq was used to map aligned reads to genes and to generate a gene count matrix. Normalized counts and differential expression analysis was performed using the DESeq2 R package. We performed gene set enrichment analysis, as described previously^{32 (link),33 (link)}.