title: GSM7999682: Accept recipient spleen, 1 week, CD8 T cell, scRNAseq; Mus musculus; RNA-Seq
library_construction_protocol: Prior to tissue collection, the kidney is perfused using a collagenase solution (2ml of 1x HBSS, 1mL of Collagenase A, and 3uL of DNase I). The kidney tissue is collected from the mouse, manually ground down, and digested in the collagenase solution. The remaining undigested tissue is manually ground down using a 70-um strainer, washed 3 times, and resuspended using FACs buffer. The spleen was collected in RPMI and then ground down using a syringe plunger of a syringe and a 70um strainer. 1 mL of ACK Lysis Buffer was added to remove red blood cells from the spleen. Cells were washed three times and resuspended in FACs Buffer. Isolated kidney or spleen cells were sorted for viable CD45+ cells via flow cytometry using an anti-CD45+ antibody and a fixable viability dye. These cells were used to construct single-cell RNA-seq libraries at Chromium 10x instrument using Chromium Next GEM Single Cell 3' kit, which were sequenced in paired-end fashion on Illumina HiSeq 2500 instrument to the depth of approximately 100 million read pairs per sample.