GSM5899221: bulk ATAC-seq data on sorted NK cells from liver of ATAC mouse replicate 2; Mus musculus; ATAC-seq
Briefly, spleen single-cell suspension was obtained by tissue disruption with glass slides. For liver, tissue was manually dissociated using a razor blade and digested with 1 mg/ml Collagenase D (Sigma Aldrich) for 30 minutes at 37°C. For salivary gland, tissue was manually dissociated using a razor blade and digested for 1 hour in 1 mg/ml Collagenase Type III (Worthington) and 4 mg/ml DNase I (Sigma-Aldrich) in HBSS at 37°C with periodic vortex. After collagenase treatment, cells isolated from the liver and salivary gland were filtered through a 70-mM cell strainer, layered in a 44% and 66% Percoll gradient (Sigma-Aldrich), and centrifuged at 1,900 g for 30 minutes without brake. For bulk ATAC-seq, 50,000 cells were sorted for each sample and frozen in FBS + 10% DMSO. Profiling of chromatin was performed by ATACseq as described previously. Briefly, 50,000 viably frozen cells were washed in cold PBS and lysed. The transposition reaction was incubated at 42°C for 45 minutes. The DNA was cleaned with the MinElute PCR Purification Kit (Qiagen catalog # 28004) and material was amplified for 5 cycles. After evaluation by real-time PCR, 8-10 additional PCR cycles were done. The final product was cleaned by aMPure XP beads (Beckman Coulter catalog # A63882) at a 1X ratio. Libraries were sequenced on a Hiseq 2500 in High Output mode in a PE50 run, using the TruSeq SBS Kit v4 (Illumina). An average of 57 million paired reads were generated per sample. For bulk RNA-seq, all cells in a given population were sorted from one mouse per sample for DP, FMSP, DN and NK cells directly into 750 mL Trizol LS. After sort, total volume was brought up to 1 mL with sterile PBS, and samples were flash frozen. Four DP, four FMSP, three DN and four NK cell samples were processed and sequenced. For RNA extraction, phase separation in cells lysed in 1 mL TRIzol Reagent (ThermoFisher catalog # 15596018) was induced with 200 mL chloroform and RNA was extracted from the aqueous phase using the miRNeasy Micro Kit (Qiagen catalog # 217084) on the QIAcube Connect (Qiagen) according to the manufacturer's protocol with 350 mL input. Samples were eluted in 15 mL RNase-free water. For transcriptome sequencing, after RiboGreen quantification and quality control by Agilent BioAnalyzer, 282-300 pg total RNA with RNA integrity numbers ranging from 7.8 to 9.4 underwent amplification using the SMART-Seq v4 Ultra Low Input RNA Kit (Clonetech catalog # 63488), with 12 cycles of amplification. Subsequently, 2.1-3 ng of amplified cDNA was used to prepare libraries with the KAPA Hyper Prep Kit (Kapa Biosystems KK8504) using 8 cycles of PCR. Samples were barcoded and run on a NovaSeq 6000 in a PE100 run, using the NovaSeq 6000 S4 Reagent Kit (200 Cycles) (Illumina). An average of 82 million paired reads were generated per sample and the percent of mRNA bases per sample ranged from 67% to 83%. Bulk RNA seq; bulk ATAC seq