GSM8751065: CC-NfL, replicate 2, scRNAseq; Mus musculus; RNA-Seq
Mice were deeply anesthetized with 2.5% avertin (2,2,2-tribromoethanol (0.5 ml/25 g body weight) and perfused with ice-cold PBS. Both hippocampi were immediately dissected and chopped into 1-2 mm2 pieces in 2 ml ice-cold Hibernate A minus calcium (HACA, BrainBits). Tissue pieces were collected by centrifuging at 300 x g for 2 min in a round-bottom 2 ml tube and added with enzyme mixes from Neural Tissue Dissociation Kit (P) (Miltenyi 130-092-628) following the manufacturer's manual with slight modifications. Each tissue sample was added into 2 ml pre-warmed Enzyme mix 1 with 5 mg/ml actinomycin D (Sigma-Aldrich A1410) and incubated on a rotator for 15 min at 37C. After adding Enzyme mix 2, samples were gently triturated with wide-orifice pipette tips, incubated for 10 min, gently triturated with regular pipette tips, incubated for another 10 min, and gently triturated with gradual addition of 10 ml ice-cold Hibernate A Low Fluorescence medium (HALF, BrainBits). Cell suspensions were filtered through 70 mm strainers (Miltenyi 130-098-462) and then centrifuged at 300 x g for 10 min at 4C. Cell pellets were carefully resuspended with wide-orifice pipette tips in HALF containing 5% FBS, with Calcein Violet AM (Thermo Fisher C34858) and propidium iodide (Thermo Fisher P1304MP) at 1 mg/ml each for live/dead FACS with BD FacsAria Fusion sorters. Sorted live single cell suspensions were collected into HALF containing 5% FBS. Cell suspensions were adjusted to 2,000 cells/ml by centrifugation at 300 x g for 10 min at 4C and resuspension in HALF containing 5% FBS. Cell viability and density were confirmed again by Trypan Blue staining and counting with Bio-Rad Tc10 counter. The single cell suspensions with viability >75% were proceeded for library preparation. Chromium Next GEM Single Cell 3ʹ GEM, Library & Gel Bead Kit v3.1 (10X Genomics PN-1000121 and PN-1000120) were used for library preparation according to the manufacturer's user guides. Cell-RT mix was prepared to aim for 8,000 cells per sample and applied to Chromium Controller for GEM generation and barcoding. Then samples were subjected to post GEM-RT cleanup, cDNA amplification (11 cycles), and library construction according to the user manual. Sample index PCR was done with 12 cycles. Libraries were then quantified by Qubit dsDNA HS Assay Kit (Thermo Fisher Q33230) and profiled by Bioanalyzer High Sensitivity DNA kit (Agilent Technologies 5067-4626).