title: GSM6194014: transplanted cells derived from unsorted progenitors, batch 2, 4 months, scRNAseq; Homo sapiens; RNA-Seq
library_construction_protocol: For in vitro differentiated samples, attached colonies (day 8-stage I) or neurospheres (day 14-stage II, day 21- stage III, day 28-stage IV, day 35-stage V) were digested using TrypLE Express Enzyme for 10 minutes at 37°C and washed twice with neural induction medium. Cells were then passed through a 35-μm cell sieve (BD) to obtain a single-cell suspension.   For graft sampels, scRNA-seq of graft samples using the 10x genomics chromium platform. 3-month-old PD mice received striatal injections of mDA progenitors derived from surface marker- tdT/AAVS1-CAG-EGFPnls-WPRE-polyA or AAVS1-CAG-EGFPnls-WPRE-polyA cell lines. Four months after transplantation, mice were deeply anesthetized, followed by transcardial perfusion with ice-cold oxygenated artificial cerebrospinal fluid (aCSF, in mM: 124 NaCl, 2.5 KCl, 1 NaH2PO4, 25 NaHCO3, 37 glucose, 2 CaCl2, 2 MgSO4). The brain was removed, 200 μm vibratome sections were collected, and the graft (most in the striatum) was microdissected under a stereo fluorescence microscope with a cooled platform. The graft pieces were dissociated using a self-made ice-cold oxygenated dissection medium (DM, in mM: 81.76 Na2SO4, 120 K2SO4, 5.8 MgCl2, 25.2 CaCl2, 1 HEPES, 20 glucose, 20 NaOH) that contained 20 units/ml papain (Worthington), 1.1 mM EDTA, 0.067 mM 2-mercaptoethanol, 5.5 mM cysteine-HCl (Sigma), and 100 units/ml DNase I (Thermo Scientific), with 30-40 minutes enzymatic digestion, followed by manual trituration using fire-polished Pasteur pipettes and filtering through a 35 μm DM-equilibrated cell strainer. Cells were then pelleted at 400g for 5 minutes. The supernatant was carefully removed. Cells were resuspended in 1-2 ml DM containing 2.5% Certified Fetal Bovine Serum (FBS, Biological Industries). Then, the debris was removed from the cell suspension using Debris Removal Solution (MiltenyiBiotec). Cell pellets were then resuspended in 200-400 μl DM containing 2.5% FBS for cell sorting, with the goal of enriching for EGFPnls-positive human cells for grafting. For in vitro differentiated samples, Chromium Single Cell 3' Reagent Kits (v2) were used for library preparation (10X Genomics).   For graft samples, Chromium Single Cell 3' Reagent Kits (v3) were used for library preparation (10X Genomics). Libraries were sequenced on an Illumina Novaseq 6000.