title: GSM6422994: mouse 3, unsort, scRNAseq; Mus musculus; RNA-Seq
library_construction_protocol: For mice without FAC-sorting, three mice that had been injected with virus were anaesthetized and then subjected to transcranial perfusion with ice-cold oxygenated self-made dissection buffer (in mM: 92 Choline chloride, 2.5 KCl, 1.2 NaH2PO4, 30 NaHCO3, 20 HEPES, 25 Glucose, 5 Sodium ascorbate, 2 Thiourea, 3 Sodium pyruvate, 10 MgSO4.7H2O, 0.5 CaCl2.2H2O, 12 N-Acetyl-L-Cysteine). The brain was removed, 300 µm vibratome sections were collected, and the PrL and IL regions were microdissected under a stereo microscope with a cooled platform. Brain slices were incubated in dissection buffer with 10 µM AMPA receptor antagonist CNQX (Abcam, Cat #ab120017) and 50 µM NMDA receptor antagonist D-AP5 (Abcam, Cat #ab120003) at 33°C for 30 min. The pieces were dissociated first using the ice-cold oxygenated dissection buffer added papain (20 units/ml, Worthington, Cat #LS003126), 0.067 mM 2-mercaptoethanol (Sigma, Cat #M6250), 1.1 mM EDTA (Invitrogen, Cat #15575020), L-Cysteine hydrochloride monohydrate (Sigma, Cat #C7880) and Deoxyribonuclease I (Sigma, Cat #D4527), with 30-40 min enzymatic digestion at 37°C, followed by 30 min 1 mg/ml protease (Sigma, Cat #P5147) and 1 mg/ml dispase (Worthington, Cat #LS02106) enzymatic digestion at 25°C. Supernatant was removed and digestion was terminated using dissection buffer containing 2% fetal bovine serum (FBS, Bioind, Cat #04-002-1A). Single-cell suspension was generated by manual trituration using fire-polishing pasteur pipettes and filtered through a 35 µm DM-equilibrated cell strainer (Falcon, Cat #352052). Cells were then pelleted at 400 g for 5 min. The supernatant was carefully removed and resuspended in 1-2 ml dissection buffer containing 2% FBS. The suspension was then subjected to the debris removal step using the Debris Removal Solution (Miltenyi, Cat #130-109-398). Cell pellets were resuspended and 48,000 cells were loaded into 3 lanes to perform 10X Genomics sequencing. For mice with FAC-sorting, PrL and IL regions were microdissected and dissociated as mice without FAC-sorting, cells were sorted to enrich for EGFP-positive retro-AAV-labeled cells. About 4893 EGFP-positive cells were captured and loaded to perform 10X Genomics sequencing. Chromium Single Cell 3' Reagent Kits (v3) were used for library preparation (10X Genomics). Libraries were sequenced on an Illumina Novaseq 6000 system.