GSM5226617: LmESAT_C7_16hr_2; Mus musculus; RNA-Seq
Splenic CD4+ T cells were enriched with the CD4 T cell negative isolation kit (Miltenyi). Cells were incubated with anti-CD16/32 in sorting buffer (2% FBS in PBS) for 10 min at 4°C to block binding to Fc receptors. Extracellular antigens were stained for 30 min at 4°C in sorting buffer. Cells were washed and resuspended in sorting buffer with SYTOX blue (Invitrogen) for exclusion of dead cells. Cells were sorted into cRPMI, pelleted and resuspended in RPMI-2% FBS. scRNA-seq of FACS-sorted cell suspensions was performed on the Chromium instrument (10X Genomics) following the user guide manual (CG00052 Rev E) and using Single Cell 3' Reagent Kit (v2). Each sample, containing approximately 8000 cells at a final dilution of 66–70 cells/µl, was encapsulated and barcoded following the manual. Viability was 82-85% for samples containing naïve cells, and 80-99% for samples containing effector cells, as confirmed with 0.2% (w/v) Trypan Blue staining. The encapsulated cells were lysed, and following reverse transcription, the barcoded cDNA was purified with DynaBeads and amplified by 14 cycles of PCR: 98 ̊C for 180 s, 12x (98 ̊C for 15 s, 67 ̊ C for 20 s, 72 ̊C for 60 s), and 72 ̊C for 60 s. 50 ng of PCR-amplified barcoded cDNA was fragmented with the reagents provided in the kit, purified with SPRI beads, and the resulting DNA library was ligated to the sequencing adapter followed by indexing PCR: 98 ̊C for 45 s; 12 cycles of (98 ̊C for 20 s, 54 ̊C for 30 s, 72 ̊C for 20 s), and 72 ̊C for 60 s. The final DNA library was double-size purified (0.6–0.8X) with SPRI beads and sequenced on Illumina Nova-Seq platform (R1 – 26 cycles, i7 – 8 cycles, R2 – 70 cycles or higher). Sequencing depth for naïve cell samples was between 65-85 million reads per sample (9.660 reads per cell), and for effector cells 175-240 million reads per sample (57.150 reads per cell).