GSM7943471: neuM22_Mock; Mus musculus; RNA-Seq
Cerebral brain tissues were dissected in a glass petri dish, minced with a scalpel, and dissociated in 5 mL Hibernate e minus calcium with 20 units/mL papain and 0.005% DNase I for 40 min at 37 °C. To promote dissociation, tissues were gently triturated through a wide-bore (~5 mm) serum-coated plastic Pasteur pipette every 10 min. After the 40 min incubation, the tissue was triturated 10 times with a serum-coated standard p1000 pipette tip and single-cell suspension was diluted with 35 mL of loading buffer. Large debris was initially removed by passing the single-cell suspension through a 70 µm MACS strainer (Miltenyi), after which cells were pelleted by centrifuging at 300 xg for 5 min at 4 °C. Debris was removed from the cell pellet using the debris removal solution from Miltenyi. Neurons were enriched from the debris-liberated cell suspension using the mouse neuron isolation kit (Miltenyi) according to manufacturer's instructions. Live single-cell suspensions were processing into sequencing libraries the Chromium Next GEM Single Cell 3ʹ Reagent Kit v3.1 (Dual Index) from 10× Genomics according to manufacturer's instructions.