title: GSM6422997: mouse 2, unsort, RNAseq projectome library; 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. Parallel PCR reactions were performed containing 50 ng of post cDNA amplification reaction cleanup material as a template. P5-Read1 (AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTC) and P7-index-Read2-EGFP (CAAGCAGAAGACGGCATACGAGATAGGATTCGGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTGgCATGGACGAGCTGTACAAG) (200 nM each) were used as primers with the NEBNext Ultra II Q5 Master Mix (NEB, Cat #M0544L). Amplification was performed using the following PCR protocol: (1) 33°C for 1 min, (2) 98° for 10 s, then 65°C for 75 s (20-24 cycles), (3) 75°C for 5 min. Reactions were re-pooled during 1X SPRI selection (Beckman, Cat #B23317), which harvested virus projection barcodes library. 431-437bp (with 120bp adaptors) libraries were sequenced using Illumina HiSeq X Ten.