GSM7792346: Tregs, CFA, rep 1; Mus musculus; RNA-Seq
Lung tissue were minced and digested with collagenase and DNase, and a single cell suspension was obtained. Mononuclear cells were isolated using a Percoll gradient centrifugation. RBCs were then lysed using a ACK lysis buffer. Tregs from FACS sorted using a FACS Melody cell sorter, and then captured for library generation and sequencing using 10X Chromium single cell capture chip according to the manufacturers protocol. The single-cell RNAseq libraries were prepared according to manufacturer's Chromium Single Cell 3ʹ protocol (10X Genomics, document # CG000204). 10,000 FACS sorted Tregs were targeted using the 10x Genomics Chromium Controller for cDNA synthesis and barcoding. cDNA quality and quantity of each sample were assessed using a Bioanalyzer High Sensitivity DNA assay. cDNA was used to begin fragmentation, followed by end-repair, adapter ligation, and sample indexing. Sample libraries were then assessed for proper construction using the same Bioanalyzer assay. Constructed libraries were pooled and quantified using a Quantabio Q cycler, and then denatured and sequenced on an Illumina Novaseq 6000 high-throughput sequencing platform with 28 cycles for the forward read and 91 cycles for the reverse read.  Each cell was sequenced to a minimum of 20,000 reads per cell per manufacturer's recommendations.