GSM4845280: NZO-3mo-M-naive-251_GT18-10984_GCCAAT_S27_L004_R1_001; Mus musculus; RNA-Seq
Data is obtained from spleen and peripheral blood lymphocytes (PBLs) of C57BL/6J and NZO/HILtJ mouse strains, hereafter B6 and NZO, respectively, at ages 3, 12, and 18 months.   Cells were stained with:   CD8 FITC, Clone 53-6.7 BD Bioscience Cat# 553031, used at 1:240 final concentration  CD3e PE, Clone 145-2C11 eBioscience (Now ThermoFisher) cat# 12-0031-85, used at 1:240 final concentration  CD62L PE-Cy7, Clone MEL-14 Tonbo Biosciences Cat# 60-0621-U100, used at 1:480 final concentration  CD44 APC-Cy7, Clone IM7 Tonbo Biosciences Cat# 25-0441-U100, used at 1:240 final concentration  and for 30 minutes at 4 degrees C. Propidium Iodide used for cell viability at 0.5ug/ml.   Then cells were sorted on a FACSAria II (BD Biosciences). Briefly, doublets were gated out, Viable PI- cells were gated, CD3e+, CD8+ cells were gated and subdivided into CD44 Low, CD62L High Naïve cells and CD44 High, CD62L+/- Memory cells. Up to 50,000 cells were sorted for ATAC-seq, remaining cells collected for other RNA preparations. 30 ng of total RNA, with the addition of 6 µl ERCC Spike-In Control Mix 1 (Ambion Thermo Fisher) diluted 1:10,000, was used for library construction. Libraries were prepared by the Genome Technologies core facility at The Jackson Laboratory using the KAPA mRNA HyperPrep Kit (KAPA Biosystems), according to the manufacturer's instructions. Briefly, the protocol entails isolation of polyA containing mRNA using oligo-dT magnetic beads, RNA fragmentation, first and second strand cDNA synthesis, ligation of Illumina-specific adapters containing a unique barcode sequence for each library, and PCR amplification.  Libraries were checked for quality and concentration using the D5000 assay on the TapeStation (Agilent Technologies) and quantitative PCR (KAPA Biosystems), according to the manufacturer's instructions.