GSM6430566: T cells, mRNA; Mus musculus; RNA-Seq
Lymphoid cells were extracted from spleens of WT, LatY136F, LatY136F x Stat6KO or LatY136F x Grap2KO mice. We isolate T cells using the EasySep Mouse T Cells Isolation Kit (StemCell). Each T cell sample was then labeled with a specific Hashtag (TotalSeq™ anti-mouse Hashtag antibody #A0301-A0310, Biolegend), a mix of 7 calibrated TotalSeq™-A anti-mouse specific antibodies (CD27, CD25, CD73, CD39, TCRb, PD-1, CD103) and a cocktail of 7 fluorescent-labeled anti-mouse antibodies (CD11b, IA/IEI, CD4, CD5, CD6, CD8a, CD90.2) plus a viability dye (Dapi) . Cells were then sorted as live singlets, CD11b– IA/IE–CD90.2+ CD6+, CD5 +. After sorting, cells were washed in PBS - 0.04% BSA as recommended in “10xGenomics sample preparation protocol” and kept on ice. Sorting of the 10 samples was performed in 3 days at 2-4 samples per day. In order to eliminate duplicates and improve cell recovery, we pooled cells sorted from 2 time points. The cells were resuspended, counted and divided into 2 wells for GEMs production. We introduced 12,000 cells per well with the expectation of obtaining 5,000 to 6,000 cells in the GEMs. Single-cell RNA-Seq libraries, HTO libraries and ADT libraries were generated using the Chromium Single Cell 3′ V3 kit (10X Genomics) according to the manufacturer instructions.