title: GSM6161341: T4 - ATAC-Seq - Tumor Trp1 CD4+; Mus musculus; ATAC-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. After flow-sorting, all samples for downstream ATAC-seq analysis were frozen in 10% DMSO and stored at −80 °C. Profiling of chromatin was performed by ATAC-seq as described by Buenrostro, et al34. Briefly, 8-50,000 viably frozen T cells were thawed, washed in cold PBS, and lysed. The transposition reaction containing TDE1 Tagment DNA Enzyme (Illumina catalog # 20034198) was incubated at 37°C for 30 minutes. The DNA was cleaned with the MinElute PCR Purification Kit (QIAGEN catalog # 28004) and material was amplified for 5 cycles using NEBNext High-Fidelity 2X PCR Master Mix (New England Biolabs catalog # M0541L). After evaluation by real-time PCR, 7-13 additional PCR cycles were done. The final product was cleaned by aMPure XP beads (Beckman Coulter catalog # A63882) at a 1X ratio, and size selection was performed at a 0.5X ratio. Libraries were sequenced on a HiSeq4000 in a PE50 run, using the HiSeq 3000/4000 SBS Kit (Illumina). An average of 57 million paired reads were generated per sample.