GSM8862750: Mo_C57B6_Control_WaterVapor; Mus musculus; RNA-Seq
Distinct nuclei isolation protocols were used in the discovery dataset and in each of the two replication datasets. For the discovery dataset, cellular nuclei were isolated from frozen dorsal striatal tissue following a 10x Genomics Demonstrated Protocol (CG000124.RevE) with modifications. One hemisphere, dorsal striatum 1 mm punch, was used for 150ul lysis buffer for nuclei extraction. To remove cellular debris and large clumps, an Iodixanol density gradient was used. An 8.5% iodixanol (OptiPrepTM Density Gradient, Millipore) buffer serves as a cushion to remove membranes at 1000xg/10 min at 4C. Clean nucleus was collected in the pellet. Propidium iodide was used to determine the nuclei concentration using a bench cytometer MoxiGoII (Orflo) and nuclei aggregation and clumping were evaluated under 60X magnification in a fluorescent microscope and brightfield. For both replication datasets, nuclei were isolated from frozen brain tissue following a published protocol with our slight modifications.82,83 Briefly, frozen brain tissue was disaggregated by pipetting up and down in chilled extraction buffer consisting of Poly(1- 22 vinylpyrrolidone-co-vinyl acetate) (Sigma #190845), 0.1% TritonX-100, and 1% bovine serum albumen (BSA) in dissociation buffer (DB; 82 mM Na2SO4, 30mM K2SO4, 10mM Glucose, 2.5mM MgCl2,10mM Hepes pH7.4, and RNase inhibitor [Protector, Roche]). Pellets containing nuclei were resuspended in DB and filtered with 70 um and 40 um strainers. To separate nuclei from debris, the nuclei suspension was mixed in a 25% (final) iodixanol layer over a 27% iodixanol cushion, followed by centrifugation (13000g x 20min, 4° C), then nuclei were washed twice in DB. Nuclei from one male mouse and one female mouse of the same ethanol treatment group were pooled. Approximately 20,000 nuclei per pooled sample were loaded into a Chromium X microfluidics controller (10x Genomics, Pleasanton, CA). 10x Genomics NextGEM 3' GEX sequencing libraries were prepared from each sample following manufacturer's instructions and sequenced at depth of 50M/sample on HiSeq4000 Sequencer. In the second replication dataset, nuclei were labeled with sample-specific oligonucleotide tag using CellPlex reagents, following a validated protocol (CG000391). Finally, nuclei were counted using a MoxiGoII (Orflo) cytometer, and the concentration was adjusted to 365 nuclei/ul in phosphate buffered saline with 2% BSA. 17,000 nuclei were loaded in each well of a Chromium Controller (10x Genomics). Nuclei from four male and four female mice were pooled in each sample. Sequencing libraries were prepared using NextGEM 3' Gene Expression reagents (10x Genomics), following manufacturer's instructions and sequenced on HiSeq4000 (Replication Dataset 1) and NovaSeq6000 (Replication Dataset 2) sequencers