GSM4848995: WT1; Mus musculus; RNA-Seq
Nine-month-old male Abi3+/+ and Abi3-/- mice were anesthetised, transcardially perfused with sterile PBS and the brain was removed. The cerebellum was removed from the right hemisphere, which was immediately finely minced on ice and transferred to a polypropylene tube containing ice-cold Accutase (Gibco, A11105-01). Tissue was incubated with rocking at 4oC for 30 minutes and then pelleted through centrifugation at 300 g for 5 minutes at 4oC. Supernatant was aspirated and samples were resuspended in ice-cold wash buffer (1X HBSS (Gibco, 14175095), 0.04% BSA (Miltenyi, 130-091-376)) and gently dissociated through pipetting. After all samples were processed, the cloudy upper suspension was transferred to a fresh tube and the remaining clumps of tissue were further dissociated using additional ice-cold wash buffer. Cell suspensions were pelleted through centrifugation at 300 g for 10 min at 4oC. The supernatant was aspirated and cells were resuspended in degassed MACS Myelin Removal Buffer (1X DPS (Gibco 14190250), 0.5% BSA (Miltenyi, 130-091-376)). Myelin removal beads (Miltenyi, 130-096-733) were added to each sample and mixed thoroughly. Samples and beads were incubated for 15 min at 4oC with rotating, washed with myelin removal buffer, then pelleted through centrifugation at 300 g for 10 min at 4oC. Supernatant was aspirated and samples were resuspended in myelin removal buffer. LS columns (Miltenyi, 130-042-401) were loaded into a QuadroMACS separator on a MACS MultiStand with Tube Rack (Miltenyi, 130-090-976 & 130-042-303 & 130-091-052). Columns were equilibrated with myelin removal buffer. Cell suspensions were loaded onto columns and myelin-depleted cell suspensions were washed through using myelin removal buffer. Cells were pelleted through centrifugation at 300 g for 5 min at 4oC. Supernatant was aspirated and cells were gently resuspended using a pipette. Cell numbers and viability were quantified using Draq7 (BD Biosciences, 564904) and Calcein-AM (BD Biosciences, 564061) staining. BD Rhapsody™ Mouse Immune Response Panel (BD, 633701 & 633753) was used for the targeted scRNA-seq library preparation. Single Cell RNA libraries were checked for quality using QuBit (ThermoFisher) and Tapestation (Agilent), then submitted to the Centre for Medical Genomics and sequenced on an Illumina NovaSeq 6000 SP (200 cycle kit) with 20% PhiX spike-in. BD Rhapsody™ Mouse Immune Response Panel (BD, 633701 & 633753)