title: GSM2902824: FGK3_tcells; Mus musculus; RNA-Seq
library_construction_protocol: Tumors for flow cytometry and RNAseq were minced in Hank's Balanced Salt Solution with 0.5mg/ml collagenase IV and 200 mg/L DNase, digested in a 37C incubator for 30 minutes and filtered to remove debris. Tumors for Single Cell Barcode Chip or intracellular cytokine stain were minced in complete RPMI with 5% serum, 0.5mg/ml collagenase IV and 200 mg/L DNase, digested in a 37C incubator for 30 minutes and filtered to remove debris. The filtrate was incubated with ACK lysis buffer (Invitrogen) for 4 minutes, washed and re-suspended in complete RPMI. For RNAseq, single-cell suspension was purified of dead cells with Lymphoprep (Stem Cell) per protocol. For RNAseq, at least three tumors were pooled for each sample collected in triplicate, with further characterization of phenotype by flow cytometry of each independent tumor. Total RNA was purified with the use of QIAzol and RNeasy Mini kit (QIAGEN), in which an on-column DNase treatment was included. Purified RNA was submitted to the Yale Center for Genomic Analysis where it was subjected to mRNA isolation and library preparation. Non-strand specific libraries were generated from 50ng total RNA using the SMARTer Ultra Low Input RNA for Illumina Sequencing kit.  Libraries were pooled, six samples per lane, and sequenced on an Illumina HiSeq 2500 (75-bp paired end reads), and aligned using STAR to the GRCm38 (mm10) reference genome. A count-based differential expression protocol was adapted for this analysis(Anders et al., 2013); mappable data were counted using HTSeq, and imported into R for differential expression analysis using the DESeq2.To find differentially regulated sets of genes for signature generation, a 1.5-Log2 fold-change difference between samples and p-adjusted (Holm-Sidak) ≤ 0.01 was used.
identifiers.namespace: GEO
identifiers.id: GSM2902824