GSM9801646: Trim28/Setdb1 Perturb-seq: lane 7, first harvest: feature-barcode capture; Mus musculus; OTHER
Tumors were dissociated to single-cell suspensions and enriched for donor T cells before sorting. Draining lymph nodes were mechanically dissociated and processed separately. Depending on experiment and sample quality, lymphocytes were enriched by Lympholyte-M density-gradient separation and/or magnetic enrichment before flow sorting. Single-cell suspensions were stained with viability dye, Fc block, and surface antibodies to identify live CD8 T cells, donor-derived transferred cells, and transduced Thy1.1+ cells, using congenic CD45.1 and CD45.2 markers together with Thy1.1 and OT-I/TCR markers where used. Purified or enriched cell suspensions from individual mice were stained with BioLegend TotalSeq-C mouse hashtag antibodies according to the manufacturer's protocol, including repeated washes to reduce ambient hashtag oligonucleotide background. Each hashtag corresponded to one mouse. Hashtag-labeled cells were pooled by tissue and harvest batch and sorted before 10x loading to maximize cell viability. Hashtag-labeled donor-cell pools were loaded onto the 10x Genomics Chromium platform, and single-cell gene-expression, CRISPR guide-capture, and feature-barcode libraries were prepared using the Chromium 5-prime workflow. The CRISPR guide/construct libraries captured sgRNA and clonal-barcode information. Guide/construct libraries were sequenced as paired-end FASTQs in which the forward read carried the cell barcode and molecular UMI and the reverse read carried the sgRNA sequence and clonal barcode flanked by known adapter sequence. Gene-expression, feature-barcode, and guide/construct libraries were sequenced on Illumina instruments.
GEO
GSM9801646_r1