GSM9686187: Pdcd1 cKO, CD8+ TIL scRNA seq; Mus musculus; RNA-Seq
Mouse splenic and thymic CD8+ T cells were isolated using CD8a+ T cell isolation kit (Miltenyi Biotec, Cat#130-104-075). Mouse splenic naïve CD8+ T cells were isolated using Naive CD8a+ T Cell Isolation Kit, mouse (Miltenyi Biotec, Cat#130-096-543). For isolating CD8+ tumor infiltrating lymphocytes (TILs), tumors were first cut into small pieces of 2-4mm and digested using tumor dissociation kit at 37°C for 45 minutes with gentle agitation (Miltenyi Biotec, Cat#130-096-730). Dead cells were further removed using Dead cell Removal kit (Miltenyi Biotec, Cat# 130-090-101). CD8+ TILs were then isolated using CD8 (TIL) microbeads, mouse (Miltenyi Biotec, Cat#130-116-478). Single cell libraries were prepared using Fluent Biosciences Pre-Templated Instant Partitions Sequencing (PIPseq) v3 Kit according to the manufacturer's protocol. Briefly, cells were first filtered through a 40-μm cell strainer to remove debris and aggregates. The viability was assessed through Trypan Blue cell counting, and samples with more than 80% viability were proceeded for sample preparation. Each sample was loaded to the PIPseq microfluidic system, which partitioned individual cells into droplets containing molecular barcodes for unique transcript identification. During reverse transcription, unique molecular identifiers (UMIs) and well-specific barcodes were incorporated to enable precise quantification and tracking of individual transcripts. Subsequently, cDNA synthesis and amplification were performed to maximize transcript coverage and minimize bias.