title: GSM6523934: E4_middle; Mus musculus; RNA-Seq
library_construction_protocol: Pooled brain tissue (n=3 per experimental group) was processed for 'glia-enriched' single cell suspensions as previously described. Briefly, mice were anesthetized via 5.0% isoflurane before exsanguination and transcardial perfusion with ice-cold Dulbecco's phosphate buffered saline (DPBS; Gibco #14040133). Following perfusion, brains were quickly removed, pooled, and whole left 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 group were immediately transferred into gentleMACS C-tube (Miltenyi #130–093-237) 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 gentleMACS Octo Dissociator instrument (Miltenyi #130–095-937) with heaters attached. After tissue digestion, cell suspensions were filtered through 70 μm mesh cell filters to remove debris following the manufacturer's suggested ABDK protocol. The resultant suspension was sequentially filtered (x2) using fresh 30 μm mesh filters. Cell viability was checked using AO/PI viability kit (Logos Biosystems # LGBD10012). All cell suspensions were determined to have > 90% viable cells. Following viability and counting, cells were diluted to achieve a concentration of ~ 1700 cells/μL in a 10μL total reaction volume. The diluted cell suspensions were loaded onto the 10X Chromium Connect automated cell portioning system. Sample libraries were constructed using Next GEM automated 3' reagents (10X Genomics, v3.1) following manufacturer's suggested protocol (#CG000286 Rev B). Final library quantification and quality check was performed using BioAnalyzer (Agilent), and sequencing performed on a NovaSeq 6000 S4 flow cell, 150 bp Paired-End sequencing (Novogene).