title: GSM7946935: Perturb_Ch1_Foxgdialout; Mus musculus; RNA-Seq
library_construction_protocol: Tissue dissociation was performed with the Papain Dissociation kit (Worthington, #LK003150) in a modification of a previously described protocol. Briefly, young mice were anesthetized then disinfected with 70% ethanol and decapitated. The brains were quickly extracted and gently dabbed with a PBS-soaked Kimwipe (Kimberly-Clark) to remove the meninge and fibroblasts. Cortices were micro-dissected in ice-cold dissection medium (Hibernate A medium (Thermo Fisher Scientific, #A1247501) with B27 supplement (Thermo Fisher Scientific, #17504044) and Trehalose (Sigma Aldrich, Cat# T9531) under a dissecting microscope. Microdissected cortices were transferred into papain solution with DNase in a cell culture dish and cut into small pieces with a razor blade. The dish was then placed onto a digital rocker in a cell culture incubator for 30 mins with rocking speed at 30 rpm at 37°C. The digested tissues were collected into a 15 mL tube and triturated with a 10 mL low bind plastic pipette 20 times and the cell suspension was carefully transferred to a new 15 mL tube. 2.7 mL of EBSS, 3 mL of reconstituted Worthington inhibitor solution, and DNase solution were added to the 15 mL tube and mixed gently. Cells were pelleted by centrifugation at 300 g for 5 mins at 4°C, followed by washing with 8ml cold dissection medium at 200g for 5 min at 4°C. Cells were resuspended in 0.5 mL ice-cold dissection medium with 10% fetal bovine serum (FBS) (Thermo Fisher Scientific, #16000069) and SYTOX dead cell stain (Invitrogen, #S34859), and subjected to FACS purification using Sony cell sorter (SH800). scRNA-seq libraries were constructed using the Chromium Next GEM Single Cell 3' Solution v3.1 kit with Feature Barcode Technology or the Chromium Next GEM Single Cell 5' Solution v2 kit with Feature Barcode Technology (10x Genomics) following the manufacturer's protocol.