GSM5862046: Normal, Early tumors, and Late tumors 1; Mus musculus; RNA-Seq
Mice were perfused with 10ml PBS, brain was harvested, and cerebellum removed. Brain was minced and resuspended in 1.5mg/ml Collagenase in HBSS with calcium and magnesium. Mixture was incubated rotating at 37°C for 30 minutes, with gentle dissociation using a P1000 pipette halfway through to break apart large pieces. Dissociated cell mixture is filtered through a 100μm filter and diluted with HBSS. All washes are pelleted at 400g for 5min. Mixture is resuspended in 30% Percoll in PBS for myelin removal at 700g for 15min with brake on low. Myelin layer is removed, and mixture is diluted and pelleted at 400g for 5min. Red blood cells are lysed using Biolegend RBC lysis buffer according to manufacturer protocol. sample was stained with CD45 and Zombie Yellow fixable viability dye. Cells were washed with RNAse free PBS with 0.1% BSA and immediately sorted on a FACs Aria Cell Sorter for viable CD45+ and CD45- populations into ice cold RNAse free PBS with 0.1% BSA. A minimum of 1 million CD45+ and CD45- cells were harvested when possible to account for the low capture rate of InDrop system. Cells were then filtered through a 70um filter before being given to the Harvard Medical School Single Cell Sequencing core where they were run on the InDrop system with the goal of encapsulating 10k cells per sample. Following encapsulation, the reverse transcription reaction was performed, and libraries were made, full protocol has been described previously. As per core recommendation, 4 libraries per 10k cells were made. Following harvest of all three samples, libraries were sequenced using the NextSeq system with 16 libraries per sequencing run, so that the depth of sequencing was calculated to be 25,000 genes per cell.