GSM7096195: Sorted_vHipp_D1_replicateD; Mus musculus; RNA-Seq
Mouse brains were collected after cervical dislocation and followed by rapid bilateral vHipp dissections from 1 mm-thick coronal brain sections using a 14G needle and frozen on dry ice. To obtain a nuclei suspension, frozen vHipp samples were homogenized in 4 mL of low-sucrose lysis buffer (0.32 M sucrose, 5 mM CaCl2, 3 mM Mg(Ace)2, 0.1 mM EDTA, 10 mM Tris-HCl) using a large clearance then a small clearance pestle of a glass dounce tissue grinder (Kimble Kontes). Homogenates were filtered through a 40 µm cell strainer (Pluriselect) into ultracentrifuge tubes (Beckman Coulter), underlaid with 5 mL of high-sucrose solution (1.8 M sucrose, 3 mM Mg(Ace)2, 1 mM DTT, 10 mM Tris-HCl) and centrifugated at 107,000 g for 1 hour at 4°C in a SW41Ti Swinging-Bucket Rotor (Beckman Coulter). Supernatant was discarded and nuclei pellets were re-suspended in 800 µL of PBS supplemented with 0.5% bovine serum albumin (BSA). DAPI was added at a 1 µg/mL final concentration. Nuclei were sorted on a BD FACS Aria II three-laser device with a 70 µm nozzle and using BD FACSDiva Software v8.0.2. Gating strategy from a representative sort are visualized in fig. S1. Briefly, debris and doublets were excluded using FSC and SSC filters, nuclei were then selected as DAPI-positive (Violet1-A laser) events, and finally GFP-positive nuclei (Blue1-A laser) were sorted directly into BSA-coated low-binding tubes. 25,000 nuclei were recovered for each sample. Following FANS, nuclei were quantified (Countess II, Life Technologies) and 8,000 per sample were loaded on a single 10X lane using Chromium Single Cell 3' Library Construction Kit (10X Genomics). cDNA libraries were prepared according to the manufacturer's protocol (10X Genomics).