GSM8443447: P1 Het, male, replicate 3, snRNAseq; Mus musculus; RNA-Seq
For E14 and P1 samples, forebrain tissue from mice were dissected and flash frozen in liquid nitrogen. The brain tissues were Dounce homogenized in ice-cold homogenization buffer (10mM Tris-HCl pH 7.4, 10mM NaCl, 3mM MgCl2, 1mM DTT, 1X cOmplete EDTA-free Protease Inhibitor (Roche 4693132001), and 0.2U/ul RNasin Inhibitor (Promega N2515) using a 2ml KIMBLE KONTES Dounce Tissue Grinder (DWK 885300-002) with 15 strokes with the “A” large clearance pestle, followed by 15 strokes of the “B” small clearance pestle. The homogenate was transferred to a 15 ml centrifuge tube. Walls of the homogenizer tubes were washed with 1ml of homogenization buffer and combined with the homogenate in the 15ml tube. The nuclei were pelleted by centrifugation in a swinging bucket rotor (Eppendorf S-4-104) at 500x g for 5 mins at 4°C. The supernatant was aspirated and discarded. The pellets were washed twice with 1ml nuclei wash buffer (DPBS, 1% BSA, and 0.2U/ul RNase inhibitor), filtered through a 40um Flowmi cell strainer (Millipore Sigma BAH136800040), and manually counted using a hemocytometer with Trypan Blue or propidium iodide (Biotium 40017).  For P21 samples, the pellets after the first centrifugation were resuspended in 1ml homogenization buffer. Density gradient centrifugation was performed to purify the nuclei from cellular debris and myelin generated during tissue dissociation. A 50% iodixanol-PBS working solution was prepared by diluting the stock solution of 60% w/v Optiprep (Millipore Sigma Aldrich D1556) with DPBS (ThermoFisher 14190136). A 35% iodixanol solution was made by diluting the 50% iodixanol with DPBS. To make the 25% Iodixanol layer, 1ml 50% iodixanol was added to 1ml of the homogenate containing the nuclei and debris. This was carefully layered on top of 2ml 35% iodixanol in a clear polycarbonate tube (Beckman Coulter 355672) and centrifuged using an Allegra 64R (Beckman Coulter 367586) at 10,000x g for 30 min at 4°C in a S0410 swinging bucket rotor (Beckman Coulter 364660) with no braking. After the centrifugation, myelin and cellular debris remaining at the top of the 25% iodixanol layer was aspirated and discarded. The purified nuclei at the interface of the 25% and 35% iodixanol layers was collected using a P1000 pipette and transferred to a clean 15ml centrifuge tube. The volume was brought up to 6 ml with nuclei wash and resuspension buffer and pelleted by centrifuging at 500x g for 5 min at 4°C. The supernatant was carefully removed, washed once with nuclei wash buffer to ensure removal of carryover ioxidanol, filtered through a 40um Flowmi cell strainer, and manually counted using a hemocytometer with Trypan Blue or propidium iodide. The libraries were made according to manufacturer's instructions for Scale Biosciences' Single Cell RNA Kit.