GSM7434375: PFC, animal 5, control, snRNAseq; Mus musculus; RNA-Seq
Micropunches were homogenized in 1.5 ml Nuclei EZ Lysis Buffer (Sigma # NUC101) supplemented with 0.2 U/ul RNAse inhibitor (NEB # ML314L) and 1X protease inhibitor (Sigma, 05892791001) in a pre-chilled 2mls KIMBLE Dounce tissue grinder (Sigma Aldrich D8938) until no tissue was visible. The lysate was filtered through a 35-µm cell strainer and centrifuged at 900 x g for 5 minutes at 4°C. The cell pellet was resuspended in wash/resuspension buffer (2% BSA in 1X PBS supplemented with 0.2 U/ul RNAse inhibitor) and mixed with Optiprep medium (60% iodixanol, Sigma-Aldrich # D1556) to a 25% final concentration. The suspension was carefully layered on to a 29% iodixanol cushion and centrifuged at 13,000 x g for 30 minutes at 4°C. The supernatant was carefully removed without disturbing the pellet and the pellet was resuspended in 100 μl wash and resuspension buffer. The nuclei were stained with DAPI and counted using a Countess Automated Cell Counter (Thermo Fisher Scientific). Single nuclei libraries were prepared using the Chromium Next GEM Single Cell 3' Kit v3.1 (10X Genomics, PN-1000128) with a target cell count of 10,000 cells. Libraries were sequenced on a NovaSeq 6000 using a S4 flow cell.  NovaSeq PE100 S4.