title: GSM6945038: Labeled stromal cells 1 HTO; Mus musculus; RNA-Seq
library_construction_protocol: 4 weeks post innoculation with cancer cells, brains were harvested from euthanized mice and processed for enzymatic tissue dissociation with the Adult Brain Dissociation kit, removal of myelin with myelin removal beads II, and removal of debris and dead cells. Single cell suspensions of each sample were incubated with an anti-human Hashtag antibody and an anti-mouse Hashtag antibody, 1:100 TruStain FeX Fc receptor blocking solution, 1:100 TruStain FeX anti-mouse CD16/32 antibody, and 1:20 APC Annexin V. Calcein Violet positive and Annexin V negative cells of each sample were sorted into 3 groups, GFP positive mCherry positive, GFP negative mCherry positive, and GFP negative mCherry negative cells. Single-cell RNA-Seq of FACS-sorted cell suspensions was performed on the Chromium instrument (10X genomics). Droplet encapsulation was performed on the cells after sorting and viability assessment. After reverse transcription and cell barcoding within droplets, emulsions were broken and cDNA purified using Dynabeads MyOne SILANE followed by PCR amplification per user manual instruction. Resulting libraries were sequenced on Illumina NovaSeq S4 platform (R1 – 28 cycles, i7 – 8 cycles, R2 – 90 cycles).