title: GSM3371685: CD45+ cells from tumor microenvironment of aPD1 treated mice; Mus musculus; RNA-Seq
library_construction_protocol: Mice were treated intraperitoneally with 200 μg anti-PD-1 (RMP1-14) or anti-CTLA-4 (9D9), used alone or in combination on days 3, 6, and 9 post-tumor transplant.  For controls, mice were injected with 200 μg of rat IgG2a isotype control antibody. Established tumors were excised from mice, minced and treated with 1 mg/ml type IA collagenase (Sigma) in HBSS (Hyclone) for 1 h at 37 °C.  Cells were washed thrice.  Red blood cells were lysed using red blood cell lysis buffer (Sigma).  To remove aggregates and clumps, cells were filtered through a 40-micron strainer. Antibodies were diluted in PBS prior to injection for a total volume of 0.5 ml per mouse. For sorting of intratumoral CD45+ cells, single cell suspensions of digested tumor were stained for 5 minutes at room temperature with 500 ng of Fc block (anti-CD16/32) and then stained with anti-CD45-PerCP/Cy5.5 (Biolegend) (1:400) plus Zombie NIR Viability dye (Biolegend) (1:500) in 100 µl of staining buffer (PBS with 2% FCS) for 30 minutes at 4 °C.  Sorting of live CD45+ cells was performed on a BD FACSAria II (BD Biosciences). The purity of the sorted cells for single cell RNA sequencing was greater than 96% as assessed during post-sort cellular analysis. Droplet-based 3′ end massively parallel single-cell RNA sequencing (scRNAseq) was performed by encapsulating sorted live CD45+ tumor infiltrating cells into droplets and libraries were prepared using Chromium Single Cell 3′ Reagent Kits v1 according to manufacturer's protocol (10x Genomics).  The generated scRNAseq libraries were sequenced using an Illumina HiSeq2500. 14x98nt read lengths were used.
attributes.tag: GEO Accession
attributes.value: GSM3371685