GSM6975622: E4; Mus musculus; RNA-Seq
Following perfusion, brains were quickly removed, and the whole right hemispheres sans brainstem and cerebellum were quickly minced using forceps on top of an ice-chilled petri dish. Minced tissue from the 3 pooled hemispheres per genotype was immediately transferred into a gentle MACS C-tube (Miltenyi #130-93-237) containing Adult Brain Dissociation Kit (ADBK) enzymatic digest reagents (Miltenyi #130–107-677) prepared according to manufacturer's protocol. Tissues were dissociated using the “37C_ABDK” protocol on the gentle-MACS Octo Dissociator instrument (Miltenyi #130-095-937) with heaters attached. After tissue digestion, cell suspensions were processed for debris removal and filtered through 70μm mesh cell filters following the manufacturer's suggested ABDK protocol. The resultant suspension was filtered sequentially two more times using fresh 30μm mesh filters. Cell viability was checked using AO/PI viability kit (Logos Biosystems # LGBD10012). Both cell suspensions were determined to have > 88% viable cells. Following viability and counting, cells were diluted to achieve a concentration of ~ 1000 cells/100uL. The diluted cell suspensions were loaded onto the 10x Genomics Chromium Controller. Each sample was loaded into a separate channel on the Single Cell 3′ Chip, and libraries were prepared using the Chromium v3 Single Cell 3′ Library and Gel Bead Kit (10x Genomics). Final library quantification and quality check were performed using BioAnalyzer (Agilent), and sequencing was performed on a NovaSeq 6000 S4 flow cell, 150 bp Paired-End sequencing (Novogene).