GSM6280797: spleen, T. gondii, 14dpi, scRNAseq; Mus musculus; RNA-Seq
Spleen tissue was manually disrupted with a razor blade in a 6cm dish and suspended in 1 mL plain PBS + 5 mM CaCl2 + 10 mg/mL B. Licheniformis protease + 125U/mL DNase / 50 mg tissue, then manually disrupted by pipette every 2 minutes for 45 minutes at 4C. Digested tissue was then mashed through a 70uM filter and washed with 10% FBS RPMI. Cells were pelleted via centrifugation at 300 x g for 5 min @ 4C. The pellet was then ACK lysed at 4C for 5 minutes, followed by washing with 10% RPMI. Library was performed according to the manufacter's instructions. Briefly, single cell drop encapsulation, RT-PCR, amplification, and adapter ligation were performed with the 10X Chromium Controller platform with the Chromium Next GEM Single Cell 3' GEM, Library and Gel Bead Kit v3.1, Chromium Next GEM Chip G Singe Cell Kit, and Single Index Kit T Set A (10X Genomics). Following encapsulation, samples were cleaned-up using Dynabeads (Invitrogen) according to manufacturer's protocol. RNA and DNA quality was checked on a Tapestation (Agilent). Paired-end sequencing was performed on a NextSeq5000 (Illumina).