GSM8722471: MAOI, TIIs; Mus musculus; RNA-Seq
Day 10 B16-OVA tumors (10 tumors were combined for each group) were harvested from experimental mice and mechanically meshed in 70-μm cell strainers to generate single cell suspensions. Single cells were washed once with T cell culture RPMI medium, resuspended in 50% Percoll, and centrifuged at 800g at 25°C for 30 min with brake off. Cell pellets enriched with TIIs were collected and sorted using a FACSAria II flow cytometer to purify live immune cells (gated as DAPI−CD45.2+ cells). Sorted TIIs were immediately delivered to the Technology Center for Genomics & Bioinformatics (TCGB) facility at UCLA for library construction and sequencing. Purified TIIs were quantified using a Cell Countess II automated cell counter (Invitrogen/Thermo Fisher Scientific). A total of 10,000 TIIs from each experimental group were loaded on the Chromium platform (10x Genomics), and libraries were constructed using the Chromium Single Cell 3′ Library & Gel Bead Kit v2 (catalog no. PN-120237, 10x Genomics) according to the manufacturer's instructions. Libraries were sequenced on an Illumina NovaSeq using a NovaSeq 6000 S2 Reagent Kit (100 cycles; catalog no. 20012862, Illumina).