Papain

Primary cultures of astrocytes were prepared from P1–P4 mouse brains. Cerebral cortices were dissected and dissociated in 15 mL tubes at 2 mg/mL papain (Worthington) in sterile Hybernate-A (Gibco) under agitation at 37 °C for 15 min. The papain was then inactivated with a mixture of ovomucoid solution and DNAase I (both Worthington). The cell suspension was then washed twice using pre-warmed Neurobasal media and centrifugation at 17×g for 5 min. The cell suspension was then strained through a 100 μm cell strainer (CellTreat), resuspended in Neurobasal medium supplemented with 10% (v/v) FBS and 10 U/mL penicillin and streptomycin (Gibco). Cells were then plated onto T-75 flasks (Falcon) pre-coated with poly-D-lysine at 5 μg/mL (Sigma) at around 10,000 cells per cm². Cells were maintained in T-75 flasks at 37 °C in a humidified 5% CO₂ chamber for 2 weeks before frozen down as stock for future cell culture experiments.

In utero electroporated mice were deeply anesthetized using isofluorane and euthanized. The brain was removed and immediately moved to pre-cooled dissociation medium (20 mM glucose, 0.8 mM kynurenic acid, 0.05 mM APV, 50 U/ml penicillin, 0.05 mg/mL streptomycin, 0.09 M Na₂SO₄, 0.03 M K₂SO₄, 0.014 M MgCl₂) on ice. Using a fluorescence stereoscope (Leica MZ10f with X-Cite Fire LED light source), the electroporated region was dissected and transferred to a new tube containing ice-cold dissociation medium. Dissociation medium was aspirated until 1 mL remained and an activated papain solution (1:1 papain [Worthington-Biochem] with 13.6 mM Cysteine-HCL, 0.002% β-mercaptoethanol, and 2.4 mM EDTA pH 8.0 in MilliQ water) was added and incubated at 37° C for 30 minutes. papain solution was removed, and tissue was washed three times and resuspended in 500 μL fresh dissociation medium. Samples were triturated 2–4 times using flame-polished borosilicate pipettes. Cell suspensions were sorted at low flow rates using a Wolf Benchtop Cell Sorter (Nanocollect Biomedical, Inc.) using red fluorescence from pCAGCells were lysed and total RNA was extracted using an AllPrep DNA/RNA Micro Kit (Qiagen). A reverse transcription reaction using a First Strand cDNA Synthesis Kit (Millipore Sigma) was used to create cDNA from the isolated RNA. The region surrounding the intended edit was amplified by PCR using primers containing universal adaptor sequences (prCR419 and prCR420). These amplicons were submitted to the Institute for Genome Sciences at the University of Maryland School of Medicine for sequencing, where samples were quantified, barcoded and sequenced on an Illumina NextSeq 550 (Illumina) according to manufacturer settings. An average of 8.9 million reads per sample were analyzed. GRCm39 was used as reference genome. Sanger sequencing of the target region from CD1 mice used in experiments were consistent with the reference genome (data not shown). Sequencing results were analyzed using CRISPResso2^{49 (link)} under prime editing mode. Default CRISPResso2 parameters were applied (quantification window 10, nicking guide sequence defined, scaffold match length 1). A contiguous quantification window was produced encompassing both pegRNA and PE3b gRNA target sequences.

Wild-type fish were processed for 10× Genomics single-cell transcriptome sequencing. Whole brains or different brain regions (Fore, OT, Hind, and sub-OT) were dissected as anatomical structures (Figure 1B, Figure 1—figure supplement 1A). Tissues were dissociated with 300 μL papain (28 units/mL, Worthington) in papain solution (1% DNase, 12 mg/mL L-cysteine in DMEM/F12), incubated at 37°C for 15 min with proper mix methods (Yu and He, 2019 (link)). Dissociated cells were washed twice with washing buffer (100 mL: 650 μL 45% glucose, 500 μL 1 M HEPES and 5 mL FBS into 93.85 mL 1× DPBS, sterilized with a 0.22 μm pore size filter), and sterilized with 40 μm cell strainers (BD Falcon) into 300–400 μL washing buffer.

Primary cultures were prepared via dissection of P1 to P3 mouse pups obtained from C57BL/6NTac WT mice or constitutive LRRK2 G2019S knock-in mice generated and maintained on the C57BL/6NTac background (Matikainen-Ankney et al., 2016 (link)) (Taconic Biosciences, NY) using methods approved by the Janelia Institutional Animal Care and Use Committee. The mesencephalic region containing the substantia nigra and ventral tegmental area was isolated stereoscopically, and the tissue was digested with papain (Worthington Biochemical, NJ) for 30 min at 37°C, gently triturated to dissociate the cells, filtered with a cell strainer, and centrifuged to remove papain and cell debris.

Different brain structures (HT, HIP, OB, RET, CX, and CB) were collected from mice (n = 7–10 mice) of 5–7 days of age immediately after sacrifice. Tissues were collected in D-PBS and were cut into small pieces of 1 mm³. Afterwards, tissues were digested using papain digestion solution containing 20 U/mL papain (ref LS003126, Worthington Biochemical Corporation, Lakewood, NJ, USA) and 200 U/mL DNase (ref D4527, Sigma Aldrich), for 1 hour at 37°C with constant rotation. This was followed by mechanical dissociation of the pellet for 3 times using a 0.02% (w/v) BSA solution (ref A4161, Sigma Aldrich) containing 333 U/mL DNase (ref D4527, Sigma Aldrich). This was followed by cell counting using a hemocytometer and centrifugation at 1000 rpm for 10 minutes. Cells were resuspended in a glia culture medium at 1000 cells/μL and plated at 200,000 cells/glass slide. Culture medium was DMEM medium (ref 41965–039, Gibco Life technologies,) supplemented with 10% fetal bovine serum (ref 16000–044, FBS-Invitrogen/Gibco), 2 mM glutamine (Sigma, G7513), 1 mM sodium pyruvate (ref 11360–039, Invitrogen/Gibco), 100 U/mL penicillin and 100 μg/mL streptomycin (Pen/Strep solution, ref 151 40–122, Invitrogen/Gibco). Cells were incubated at 37°C with 5% CO₂ for 8 days and medium was changed every 3 days.