GSM9156486: LCK Project (GEX); Mus musculus; RNA-Seq
Splenocytes were collected on day 5 post LM-OVA-immunization. Samples were enriched for CD8+ cells by using Dynabeads™ Untouched™ Mouse CD8 Cells Kit (Cat#11417D, Invotrogen) according to manufacturer's instructions. The cells were resuspended in HBSS and stained with with LIVE/DEAD near-IR dye, anti-CD8a BV421, anti-CD45.1 PE, and anti-CD45.2 APC antibodies together with TotalSeq-C Hashtag antibodies for multiplexing (BioLegend; MH1: #155861, MH2: #155863, MH3: #155865, MH5: #155869, MH6: #155871, MH7: #155873, MH8: #155875, MH9: #155877). Viable CD8a+, CD45.2+OT-I cells were FACS sorted. The viability and concentration of cells after sort were measured using the TC20 Automated Cell Counter (1450102, Bio-Rad). The viability of the cells pre-loading was 85%. Cells were loaded onto a 10X Chromium machine (10X Genomics) aiming at the yield of X cells per sample The sequencing libraries were prepared using Chromium Next GEM Single Cell 5' Reagent Kits v2 with Feature Barcode technology for CRISPR Screening and Cell Surface Protein (10X Genomics, #PN-1000263, #PN-1000286, #PN-1000541, #PN-1000190, #PN-1000215, #PN-1000250) according to the protocol provided by the manufacturer (#CG000511 Rev C).