GSM9230082: GL261, ICB, mixed pooled of mice 9 and 10; Mus musculus; RNA-Seq
Tumors were harvested at day 21 p.i. and tumor infiltrating leukocytes were sorted (CD45+ DRAQ7-). From four individual mice from each treatment group, cells were suspended in Stain buffer (BD Pharmigen) at a density of 1 million per 100μL and stained with anti-mouse CD16/CD32 Fc Block (BD Pharmigen) for 10 min. Next, an anti-mouse CD45 antibody (Clone 30-F11, BD Pharmigen) was added, and cells were incubated for 20 min at 4 °C, washed with PBS, and sorted in PBS 1% BSA. Sorting was performed on a MoFlo Astrios (Beckman Coulter). Directly after sorting, cell quantity and viability of CD45+DRAQ7- cells were measured, and two pools were prepared from each treatment group comprised of 5000 cells from each of two individual mice. Each cell suspension was loaded on a 10× Genomics Chromium instrument. Single-cell RNA-Seq libraries were prepared using Chromium Single Cell 3′ v3.1 Reagent Kit according to manufacturer's protocol. Library quantification and quality assessment was performed using a Qubit fluorometer and a Tapestation (DNA High sensitivity chip, Agilent Technologies). Libraries were sequenced on an Illumina NovaSeq 6000 using paired-end 28 × 90 bp as sequencing mode.