SRX18471357 GSM6779214_r1 SRP411070 PRJNA907814 SRS15945096 GSM6779214 GSM6779214 RNA-Seq GENOMIC SINGLE CELL cDNA Human cortical cells (frozen at day 25) were thawed and plated on Matrigel-coated plates using DDM/B27+Nb/B27 medium at 37˚C with 5% CO 2 . Seven days after thawing (day 32), the cells were dissociated using Accutase and plated on Matrigel-coated plate at high confluency (450,000-700,000 cells/cm 2 ) with LV-NeuroD1 promoter-CreERT2-WPRE and LV-CAG-DIO-EGFP-T2A-tCD8-WPRE. Four days later (day 36), the medium was changed to DDM/B27+Nb/B27 medium with 1µM 4-OHT and 10µM DAPT (Abcam, Cat#ab120633). Two days after 4-OHT/DAPT induction (day 38), the medium was changed to fresh DDM/B27+Nb/B27 medium. At day 40, cortical cells were dissociated using NeuroCult Enzymatic Dissociation Kit for Adult CNS Tissue (STEMCELL technologies, Cat#05715) following the manufacturer's instructions. For CD8+ magnetic cell sorting (MACS), dissociated cells were incubated with magnetic beads conjugated anti-human CD8 (Miltenyi Biotec, Cat#130-045-201) in MACS buffer, mixture of autoMACS Rinsing Solution (Miltenyi Biotec, Cat#130-091-222) and MACS BSA Stock Solution (Miltenyi Biotec, Cat#130-091-376), at 4˚C for 10 min. CD8 positive selection was carried out with MS or LS columns (Miltenyi Biotec, Cat# 130-042- 201 or 130-042-401) according to the manufacturer's instructions. The sorted cells were plated on mouse astrocyte-coated plates at intermediate confluency (160,000 cells/cm 2 ). The culture medium was changed two times a week. At indicated time points, cortical neurons were dissociated using NeuroCult Enzymatic Dissociation Kit following the manufacturer's instructions and resuspended in PBS containing 1%BSA. The cell suspension was passed through a cell strainer (Merk, Cat#BAH136800040) and counted using a LUNA dual fluorescence cell counter (Logos Biosystems) after Acridine Orange/Propidium Iodide staining (Logos Biosystems, Cat#F23001). Library preparations for the scRNA-seq was performed using 10X Genomics Chromium Single Cell 3' Kit, v3.1 NextGEM chemistry (10X Genomics, Pleasanton, CA, USA). The cell count and the viability of the samples were accessed using LUNA dual florescence cell counter and a targeted cell recovery of 10,000 cells was aimed for each of the samples. Following cell count and QC, the samples were immediately loaded onto the Chromium Controller. Single cell RNAseq libraries were prepared using manufacturer's recommendations (Single cell 3' reagent kits v3.1 user guide; CG000204 Rev D), and at different check points the library quality was assessed using Qubit (Thermo Fisher Scientific) and Bioanalyzer (Agilent). With a sequencing saturation targeted for ~ 40-60%, single cell libraries were sequenced on Illumina's NovaSeq 6000 platform using paired-end sequencing workflow and with recommended 10X; v3.1 read parameters (28-8-0-91 cycles). Single-nuclei libraries were generated using the 10x Chromium Single-Cell Instrument and Chromium Single Cell 3' Reagent v3 Kits (10x Genomics) according to the manufacturer's protocol. In brief, the single mouse liver nuclei suspension was loaded in the Chromium chip B for partitioning into nanoliter-scale Gel Beads-in-emulsion (GEMs). After GEMs generation, the obtained emulsion was incubated in a C1000 Touch Thermal Cycler (Bio-Rad) under the following program: 53°C for 45 min, 85°C for 5 min and hold at 4°C. Incubation of the GEMs produced barcoded, full-length cDNA from poly-adenylated mRNA. After incubation, single-cell droplets were dissolved, and full-length cDNA were isolated using Cleanup Mix containing Silane Dynabeads. To generate sufficient mass for library construction, the cDNA was amplified via PCR: 98°C for 3 min; 12 cycles of 98°C for 15 s, 63°C for 20 s, 72°C for 1 min; 72°C for 1 min; and hold at 4°C. Subsequently, the amplified cDNA was fragmented, end- repaired, A-tailed and index adaptor ligated, with SPRIselect cleanup in between steps. The final gene expression library was amplified by PCR: 98°C for 45 s; 10-12 cycles of 98°C for 20 s, 54°C for 30 s, 72°C for 20 s. 72°C for 1 min; and hold at 4°C. The sequencing-ready libraries were cleaned up with SPRIselect beads. Before sequencing, the fragment size of every library was analyzed using the Bioanalyzer high-sensitivity chip and were sequenced on HiSeq2500 or NovaSeq6000 instruments with the following sequencing parameters: 28 bp read 1 – 10 bp index 1 (i7) – 10 bp index 2 (i5) – 75 bp read 2. Single-cell RNA-seq (10X Genomics) Illumina NovaSeq 6000 SRX18471358 GSM6779215_r1 SRP411070 PRJNA907814 SRS15945097 GSM6779215 GSM6779215 RNA-Seq GENOMIC SINGLE CELL cDNA Human cortical cells (frozen at day 25) were thawed and plated on Matrigel-coated plates using DDM/B27+Nb/B27 medium at 37˚C with 5% CO 2 . Seven days after thawing (day 32), the cells were dissociated using Accutase and plated on Matrigel-coated plate at high confluency (450,000-700,000 cells/cm 2 ) with LV-NeuroD1 promoter-CreERT2-WPRE and LV-CAG-DIO-EGFP-T2A-tCD8-WPRE. Four days later (day 36), the medium was changed to DDM/B27+Nb/B27 medium with 1µM 4-OHT and 10µM DAPT (Abcam, Cat#ab120633). Two days after 4-OHT/DAPT induction (day 38), the medium was changed to fresh DDM/B27+Nb/B27 medium. At day 40, cortical cells were dissociated using NeuroCult Enzymatic Dissociation Kit for Adult CNS Tissue (STEMCELL technologies, Cat#05715) following the manufacturer's instructions. For CD8+ magnetic cell sorting (MACS), dissociated cells were incubated with magnetic beads conjugated anti-human CD8 (Miltenyi Biotec, Cat#130-045-201) in MACS buffer, mixture of autoMACS Rinsing Solution (Miltenyi Biotec, Cat#130-091-222) and MACS BSA Stock Solution (Miltenyi Biotec, Cat#130-091-376), at 4˚C for 10 min. CD8 positive selection was carried out with MS or LS columns (Miltenyi Biotec, Cat# 130-042- 201 or 130-042-401) according to the manufacturer's instructions. The sorted cells were plated on mouse astrocyte-coated plates at intermediate confluency (160,000 cells/cm 2 ). The culture medium was changed two times a week. At indicated time points, cortical neurons were dissociated using NeuroCult Enzymatic Dissociation Kit following the manufacturer's instructions and resuspended in PBS containing 1%BSA. The cell suspension was passed through a cell strainer (Merk, Cat#BAH136800040) and counted using a LUNA dual fluorescence cell counter (Logos Biosystems) after Acridine Orange/Propidium Iodide staining (Logos Biosystems, Cat#F23001). Library preparations for the scRNA-seq was performed using 10X Genomics Chromium Single Cell 3' Kit, v3.1 NextGEM chemistry (10X Genomics, Pleasanton, CA, USA). The cell count and the viability of the samples were accessed using LUNA dual florescence cell counter and a targeted cell recovery of 10,000 cells was aimed for each of the samples. Following cell count and QC, the samples were immediately loaded onto the Chromium Controller. Single cell RNAseq libraries were prepared using manufacturer's recommendations (Single cell 3' reagent kits v3.1 user guide; CG000204 Rev D), and at different check points the library quality was assessed using Qubit (Thermo Fisher Scientific) and Bioanalyzer (Agilent). With a sequencing saturation targeted for ~ 40-60%, single cell libraries were sequenced on Illumina's NovaSeq 6000 platform using paired-end sequencing workflow and with recommended 10X; v3.1 read parameters (28-8-0-91 cycles). Single-nuclei libraries were generated using the 10x Chromium Single-Cell Instrument and Chromium Single Cell 3' Reagent v3 Kits (10x Genomics) according to the manufacturer's protocol. In brief, the single mouse liver nuclei suspension was loaded in the Chromium chip B for partitioning into nanoliter-scale Gel Beads-in-emulsion (GEMs). After GEMs generation, the obtained emulsion was incubated in a C1000 Touch Thermal Cycler (Bio-Rad) under the following program: 53°C for 45 min, 85°C for 5 min and hold at 4°C. Incubation of the GEMs produced barcoded, full-length cDNA from poly-adenylated mRNA. After incubation, single-cell droplets were dissolved, and full-length cDNA were isolated using Cleanup Mix containing Silane Dynabeads. To generate sufficient mass for library construction, the cDNA was amplified via PCR: 98°C for 3 min; 12 cycles of 98°C for 15 s, 63°C for 20 s, 72°C for 1 min; 72°C for 1 min; and hold at 4°C. Subsequently, the amplified cDNA was fragmented, end- repaired, A-tailed and index adaptor ligated, with SPRIselect cleanup in between steps. The final gene expression library was amplified by PCR: 98°C for 45 s; 10-12 cycles of 98°C for 20 s, 54°C for 30 s, 72°C for 20 s. 72°C for 1 min; and hold at 4°C. The sequencing-ready libraries were cleaned up with SPRIselect beads. Before sequencing, the fragment size of every library was analyzed using the Bioanalyzer high-sensitivity chip and were sequenced on HiSeq2500 or NovaSeq6000 instruments with the following sequencing parameters: 28 bp read 1 – 10 bp index 1 (i7) – 10 bp index 2 (i5) – 75 bp read 2. Single-cell RNA-seq (10X Genomics) Illumina NovaSeq 6000 SRX18471359 GSM6779216_r1 SRP411070 PRJNA907814 SRS15945098 GSM6779216 GSM6779216 RNA-Seq GENOMIC SINGLE CELL cDNA Human cortical cells (frozen at day 25) were thawed and plated on Matrigel-coated plates using DDM/B27+Nb/B27 medium at 37˚C with 5% CO 2 . Seven days after thawing (day 32), the cells were dissociated using Accutase and plated on Matrigel-coated plate at high confluency (450,000-700,000 cells/cm 2 ) with LV-NeuroD1 promoter-CreERT2-WPRE and LV-CAG-DIO-EGFP-T2A-tCD8-WPRE. Four days later (day 36), the medium was changed to DDM/B27+Nb/B27 medium with 1µM 4-OHT and 10µM DAPT (Abcam, Cat#ab120633). Two days after 4-OHT/DAPT induction (day 38), the medium was changed to fresh DDM/B27+Nb/B27 medium. At day 40, cortical cells were dissociated using NeuroCult Enzymatic Dissociation Kit for Adult CNS Tissue (STEMCELL technologies, Cat#05715) following the manufacturer's instructions. For CD8+ magnetic cell sorting (MACS), dissociated cells were incubated with magnetic beads conjugated anti-human CD8 (Miltenyi Biotec, Cat#130-045-201) in MACS buffer, mixture of autoMACS Rinsing Solution (Miltenyi Biotec, Cat#130-091-222) and MACS BSA Stock Solution (Miltenyi Biotec, Cat#130-091-376), at 4˚C for 10 min. CD8 positive selection was carried out with MS or LS columns (Miltenyi Biotec, Cat# 130-042- 201 or 130-042-401) according to the manufacturer's instructions. The sorted cells were plated on mouse astrocyte-coated plates at intermediate confluency (160,000 cells/cm 2 ). The culture medium was changed two times a week. At indicated time points, cortical neurons were dissociated using NeuroCult Enzymatic Dissociation Kit following the manufacturer's instructions and resuspended in PBS containing 1%BSA. The cell suspension was passed through a cell strainer (Merk, Cat#BAH136800040) and counted using a LUNA dual fluorescence cell counter (Logos Biosystems) after Acridine Orange/Propidium Iodide staining (Logos Biosystems, Cat#F23001). Library preparations for the scRNA-seq was performed using 10X Genomics Chromium Single Cell 3' Kit, v3.1 NextGEM chemistry (10X Genomics, Pleasanton, CA, USA). The cell count and the viability of the samples were accessed using LUNA dual florescence cell counter and a targeted cell recovery of 10,000 cells was aimed for each of the samples. Following cell count and QC, the samples were immediately loaded onto the Chromium Controller. Single cell RNAseq libraries were prepared using manufacturer's recommendations (Single cell 3' reagent kits v3.1 user guide; CG000204 Rev D), and at different check points the library quality was assessed using Qubit (Thermo Fisher Scientific) and Bioanalyzer (Agilent). With a sequencing saturation targeted for ~ 40-60%, single cell libraries were sequenced on Illumina's NovaSeq 6000 platform using paired-end sequencing workflow and with recommended 10X; v3.1 read parameters (28-8-0-91 cycles). Single-nuclei libraries were generated using the 10x Chromium Single-Cell Instrument and Chromium Single Cell 3' Reagent v3 Kits (10x Genomics) according to the manufacturer's protocol. In brief, the single mouse liver nuclei suspension was loaded in the Chromium chip B for partitioning into nanoliter-scale Gel Beads-in-emulsion (GEMs). After GEMs generation, the obtained emulsion was incubated in a C1000 Touch Thermal Cycler (Bio-Rad) under the following program: 53°C for 45 min, 85°C for 5 min and hold at 4°C. Incubation of the GEMs produced barcoded, full-length cDNA from poly-adenylated mRNA. After incubation, single-cell droplets were dissolved, and full-length cDNA were isolated using Cleanup Mix containing Silane Dynabeads. To generate sufficient mass for library construction, the cDNA was amplified via PCR: 98°C for 3 min; 12 cycles of 98°C for 15 s, 63°C for 20 s, 72°C for 1 min; 72°C for 1 min; and hold at 4°C. Subsequently, the amplified cDNA was fragmented, end- repaired, A-tailed and index adaptor ligated, with SPRIselect cleanup in between steps. The final gene expression library was amplified by PCR: 98°C for 45 s; 10-12 cycles of 98°C for 20 s, 54°C for 30 s, 72°C for 20 s. 72°C for 1 min; and hold at 4°C. The sequencing-ready libraries were cleaned up with SPRIselect beads. Before sequencing, the fragment size of every library was analyzed using the Bioanalyzer high-sensitivity chip and were sequenced on HiSeq2500 or NovaSeq6000 instruments with the following sequencing parameters: 28 bp read 1 – 10 bp index 1 (i7) – 10 bp index 2 (i5) – 75 bp read 2. Single-cell RNA-seq (10X Genomics) Illumina NovaSeq 6000