GSM8062059: Brain, wildtype female 2; Mus musculus; RNA-Seq
Each mouse was anesthetised, transcardially perfused with sterile cold PBS, and then the brain was collected. After removing the cerebellum and olfactory bulb, the right hemisphere was immediately finely minced on ice and transferred to a polypropylene tube containing 10 ml 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 5 ml ice-cold wash buffer [1X HBSS (Gibco, 14175095), 0.04% BSA (Miltenyi, 130-091-376)] and gently dissociated through trituration using a 5 ml serological pipette. 70 µm and 40 µm cell-strainers (Corning, 352350 & 352340) were moistened with 5 ml ice-cold wash buffer and samples were sequentially strained through into fresh polypropylene tubes, each filter was rinsed with an additional 5 ml of 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 1080 µl of degassed MACS Myelin Removal Buffer (1X DPS (Gibco 14190250), 0.5% BSA (Miltenyi, 130-091-376)) and transferred to 2 ml DNA LoBind tubes (Eppendorf, 022431048). 120 µl of myelin removal beads (Miltenyi, 130-096-733) were added to each sample and mixed thoroughly by pipetting. Samples and beads were incubated for 15 min at 4oC with rotating. Samples were transferred to 15 ml polypropylene tubes and washed with 10 ml myelin removal buffer, then pelleted through centrifugation at 300 g for 10 min at 4oC. Supernatant was aspirated and samples were resuspended in 2ml 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 three successive 1 ml volumes of myelin removal buffer which were collected in 15 ml polypropylene tubes. Cell suspensions were loaded onto columns and myelin-depleted cell suspensions were washed through using four successive 1 ml volumes of myelin removal buffer. Effluent was made up to 10 ml total volume and cells were pelleted through centrifugation at 300 g for 5 min at 4oC. Supernatant was aspirated to a residual volume of 200 µl and cells were gently resuspended using a pipette. Cells were then pelleted through centrifugation at 300 g for 5 min at 4oC before resuspension in 200 µl resuspension buffer (1X DPS (Gibco 14190250); 0.04% BSA (Miltenyi, 130-091-376)), another centrifugation step using the same settings and finally resuspension in 50 µl resuspension buffer. An aliquot of cells was stained with trypan blue, and the cell concentration and viability were determined by manual counting using a disposable hemocytometer slide (C-Chip, SKC, Inc., Covington, GA) and the 40X objective of an EVOS XL Core microscope Single cell suspensions were mixed with single cell master mix containing lysis buffer and reverse transcription reagents, following the Chromium Next GEM Single Cell 3' Reagent Kits User v3.1 (Dual Index) Guide, CG000315 Rev A (10X Genomics, Pleasanton, CA). Each single cell mix was then loaded into a single well of a Single Cell Chip G and run on the Chromium Controller for GEM generation and barcoding. Sample processing and library preparation were performed according to the manufacturer's instructions using the Chromium Next GEM Single Cell 3' v3.1 dual index kit (10X Genomics, Inc.) and SPRIselect paramagnetic bead-based chemistry (Beckman Coulter Life Sciences, Indianapolis, IN). The cDNA and library quality were assessed using the Agilent 2100 Bioanalyzer and a High Sensitivity DNA kit (Agilent Technologies, Santa Clara, CA) and the final library concentration was determined using a Qubit Fluorometer and the dsDNA HS assay kit (Thermo Fisher Scientific, Waltham, MA). Sequencing was carried out on a NovaSeq 6000 v1.5 S2 100 cycle chip (Illumina) with 28-10-10-91 read setup.