GSM8178732: Normal, DMGMOUSEWT, scRNASeq; Mus musculus; RNA-Seq
Three weeks after injection, untreated mice were euthanized for brainstem tumor single-cell isolation and Next-generation sequencing. Before euthanasia, mice were anesthetized with 2% isofluorane and perfused for 8 minutes with cold saline (1X PBS) and/or until the liver turned pale. Upon dissection of the brainstem, the tissue was placed in isolation media consisting of RPMI 1640 with GlutaMAX Supplement and HEPES (Gibco) with 0.01% 50X B27 supplement (Gibco). The tissue was then homogenized in a 15ml glass tissue grinder, filtered through a 70μm filter and spun down at 300g for 10 minutes at 4 degrees. The pellet was resuspended in 1ml of isolation media (per brainstem), 100ul of myelin removal beads were added (Miltenyi Biotec) and the mixture was incubated for 15 minutes. After one wash, the solution was then passed through LS+ positive separation columns (Miltenyi Biotec) to deplete the myelin. After another wash with single-cell buffer (HBBS + 0.04% BSA), the pellet was resuspended in 400 ul of RBC lysis buffer and incubated on ice for 2 minutes. Then, 1ml of single-cell buffer was added and sample was spun down at 250g for 5 minutes. Three more washes with single-cell buffer were performed and the final pellet was resuspended in the appropriate volume of single-cell buffer to have an approximate concentration of 1x106 cells/mL. Cell counting and viability was assessed with Countess 3FL Automated Cell Counter. Dissociated cells were processed for single-cell gene expression capture (scRNA-seq) using the 10X Chromium 3' Library and Gel Bead Kit (10x Genomics), following the manufacturer's user guide. After Gel bead-in-EMulsion Reverse Transcription (GEM-RT) reaction, 12-15 cycles of polymerase chain reaction (PCR) amplification were performed to obtain cDNAs used for RNA seq library generation Libraries were prepared following the manufacturer's user guide and sequenced on the Illumina NovaSeq 6000 Sequencing System at the Columbia University Single Cell Analysis Core