GSM8816819: wildtype C57/BL6 distal colonic tumor from mouse 4; Mus musculus; RNA-Seq
Mouse tumors were incubated rotating at 4˚ C for 1.25 h in a chelating buffer of Dulbecco's PBS (DPBS) with 20mM HEPES, 3mM EDTA, and 0.5mM DTT, then gently washed in DPBS. Tumors were then resuspended in DPBS with 5mg/ml cold active protease and 2.5mg/ml DNase and incubated at 4˚ C for 25 minutes with rotation. Tissues were then pipetted 10-20 times with a pipette to liberate single cells, which were passed through a 70μm filter into a new tube for washes. Cells were washed in DPBS with 0.02% BSA before proceeding immediately to scRNA-seq. scRNA sequencing was performed using Pipseq T2 V4plus kits from Fluent Biosciences. In summary, cells were co-encapsulated with capture beads through vortexing. The resulting emulsion was then incubated to allow for lysis of cells and hybridization of mRNA to barcoded oligos on the beads. Beads were released from droplets and washed, before undergoing reverse transcription and PCR reactions, resulting in amplified barcoded transcript libraries. Libraries were then fragmented, a-tailed and indexed for sequencing. Sequencing was erformed on Novaseq XP, targeting 100M reads per sample. scRNA-seq was performed on tumors isolated from two WT mice and three DPEP1 KO mice.