GSM9008870: KO GEX library; Mus musculus; OTHER
We first sorted 5x10^4 P14 T cells from the IEL, liver, and spleen of 4 pooled mice per group (control vs NEURL3 KO, control vs RNF149 KO, and control vs WSB1 KO) into DMEM supplemented with 2% bovine growth serum and 10 mM HEPES. Due to the number of groups, all control samples were pooled together and all KO samples were pooled together. Following the first sort, hash-tagged cells were pooled incubated with antibody-derived tags (ADT, TotalSeq-C Mouse Universal Cocktail, V1.0) and washed thoroughly. For the second sort, all stained cells from the pooled tissues were sorted as well as 5x10^4 CD8+ T cells from a naive spleen were also sorted to allow for batch correction with totalVI. scRNA-seq was performed using 10X Genomics 5'v2 with feature barcoding for cell surface protein and immune receptor mapping following the manufacturer's instructions. After cDNA amplification, the smaller fragments containing the TotalSeq-C–derived cDNA were purified and saved for feature barcode library construction. The larger fragments containing transcript-derived cDNA were saved for gene expression library construction. For both cDNA portions, the library size was measured with an Agilent Bioanalyzer D5000 high-sensitivity DNA assay and quantified using a Qubit 2.0 fluorometer. Transcript-derived cDNA was processed into RNA libraries. After enzymatic fragmentation and size selection, purified cDNA was ligated to an Illumina R2 sequence and indexed with unique Dual Index TT set A index sequences with an index PCR. TotalSeq-C–derived cDNA was processed into feature barcode libraries. The cDNA was indexed with unique Dual Index TN set A index sequences with an index PCR. The two libraries were pooled together on the basis of molarity with relative proportions of 40% GEX (RNA) and 10% surface (feature barcode) for each control and KO pooled samples. This pool was then sequenced on an Illumina NovaSeq X Plus 10B. The sequencing configuration followed 10X Genomics specifications.