title: GSM6161332: P7 - Tumor Pmel CD8+CD4+; Mus musculus; RNA-Seq
library_construction_protocol: Samples were isolated from 3 independent experiments of pooled spleens (n=3) or tumors (n=5-8) as follows: (i)Naïve TRP1 CD4+ T cells and naïve PMEL CD8+ T cells were sorted by flow cytometry from spleens of Trp1 and Pmel-1 transgenic mice. (ii) TRP1 CD4+ and TRP1 CD4+CD8+ were sorted from B16 tumors of C57BL/6 mice that had been adoptively transferred with naïve TRP1 CD4+ T cells 11 days prior. (iii) PMEL CD8+ and PMEL CD4+CD8+ were sorted from B16 tumors of C57BL/6 mice that had been adoptively transferred with naïve PMEL CD8+ T cells 11 days prior. Cells were gated on congenic labels (TRP1 – CD45.1, PMEL - CD90.1), CD3+, CD4+CD8- (TRP1), CD4+CD8+ (TRP1 and PMEL), or CD4-CD8+ (PMEL). Samples were directly sorted into Trizol (Invitrogen), adjusted to 500uL total volume and frozen and stored at −80 °C. RNA was extracted using RNeasy mini kit (Qiagen) per instructions provided by the manufacturer. After ribogreen quantification and quality control of Agilent BioAnalyzer, total RNA underwent amplification using the SMART-seq V4 (Clonetech) ultralow input RNA kit for sequencing (12 cycles of amplification for 2–10 ng of total RNA). Subsequently, 10 ng of amplified cDNA was used to prepare Illumina Hiseq libraries with the Kapa DNA library preparation chemistry (Kapa Biosystems). Samples were run on a Hiseq 4000, in a 50-bp/50-bp paired-end run, using the TruSeq SBS Kit v3 (Illumina).