SRX23428310 GSM8042313_r1 SRP486498 PRJNA1070591 SRS20282770 GSM8042313 GSM8042313 RNA-Seq TRANSCRIPTOMIC SINGLE CELL cDNA For the scRNAseq, Neocortices of P90 Emx/tdTom (control, n=4) and Emx/Tom/DTA (ablated, n=2) were microdissected and the tissue dissociated into single-cell suspensions, as previously described. Briefly, mice were transcardially perfused with ice-cold oxygenated artificial cerebrospinal fluid (22 mM NaCl, 0.63 mM KCl, 0.4 mM NaH2PO4 * 2H2O, 6.5 mM NaHCO3, 25 mM Saccharose, 5 mM Glucose, 0.5 mM CaCl2, 4 mM MgSO4. pH 7.3) and the brains collected. The tissue was sectioned at the vibratome in ice-cold artificial cerebrospinal fluid and the motor cortex and cingulate cortex were microdissected. Tissue dissociation was performed with the Adult Brain Dissociation Kit (Miltenyi Biotec) following manufacturer'sinstructions(red blood cells removal step was not included). After debris removal, the cells were resuspended in ice-cold 1% BSA in artificial cerebrospinal fluid, filtered with 30 μm filter (Sysmex Partec) and FACS sorted with the BD Influx System (USB. BD FACS™) to separate GFP+ and TdTom+OL lineage cells. A scRNA-Seq experiment set with a cohort of control and experimental mice was discarded due to strong batch effects, most likely due to stress during the FACS procedure For the snRNAseq, to assess the impact of dOPC ablation immediately when ablation occurs, cortical tissue was analysed unsing single-nucleus RNA sequencing (snRNA-seq) of antibody-hashed samples. Neonatal mice were sacrificed on the day of birth by decapitation, then the motor cortex region was dissected in icecold oxygenated cutting solution containing 87mM NaCl, 2.5mM KCl, 1.25mM NaH2PO4, 26mM NaHCO3, 0.5mM CaCl2, 4mM MgSO4, 75mM sucrose, and 20mM glucose, before samples were snapfrozen on dry ice in a sterile microtube. The samples were stored at -80°C until further processed. To isolate nuclei, tissue fragments from two animals with identical genotype were combined in nuclei isolation buffer (250mM sucrose, 25mM KCl, 5mM MgCl2, 10mM Tris buffer pH 8.0, 0.1% NP40, 0.1mM DTT and 0.4U/μl RNAse inhibitors), and gently homogenized with a plastic pestle on ice. Subsequently, the homogenate was filtered using a 40μm strainer, and the nuclear pellet was collected upon centrifugation. The pellet was then washed twice with Nuclei Wash and Resuspension buffer (NWR) containing 2% BSA in PBS and 0.4U/μl RNAse inhibitors. Finally, the nuclei were resuspended in ST Staining Buffer (ST-SB: 2% BSA, 0.02% Tween-20, 10mM Tris pH 7.5, 146mM NaCl, 1mM CaCl2, and 21mM MgCl2), filtered again with 40μm strainer and collected for inspection and counting. Hashtag antibody tagging was done as described previously with minor adjustments. Two sets of ablated and two sets of control nuclear samples (1M nuclei per 100μl of ST-SB) were first blocked with TruStain FcX™ (anti-mouse CD16/32, Biolegend #101319) before the addition of 1μg/100μl Mab414 nuclei hashing antibodies (TotalSeq™ A0451-54, Biolegend #682205, #682207, #682209, #682211). To remove the unbound antibodies, samples were washed twice with ST Wash Buffer (10mM Tris, 146mM NaCl, 1mM CaCl2, 21mM MgCl2, 2% BSA, 0.4U/μL of RNAse inhibitor) and again with NWR buffer to avoid the detergent and magnesium carry-over prior to sequencing For the scRNAseq, the sorted cells were processed with the Chromium Single Cell A Chip kit v2 and library prep with the Chromium Single Cell 3´Library & Gel Beads kit v2 (10X Genomics) according to the manufacturer's instructions. A total of 3,000 cells for each sample were loaded on the Chromium Single Cell A Chip, although a lower number of cells was recovered in singlet and passed the quality control. For the snRNAseq, the nuclei were resuspended in NWR, strained with a 20μm and 10μm strainer before counting, sample pooling and downstream processing with 10X platform for single-nuclei sequencing For the snRNAseq, 4 hastag antibodies were used to label our samples, from Biolegend TotalSeq™ A0451-54. A0451 (DTA) TTCCTGCCATTACTA, A0452 (DTA) CCGTACCTCATTGTT, A0453 (Ctrl) GGTAGATGTCCTCAG, A0454 (Ctrl) TGGTGTCATTCTTGA Illumina NovaSeq 6000 SRX23428311 GSM8042314_r1 SRP486498 PRJNA1070591 SRS20282771 GSM8042314 GSM8042314 RNA-Seq TRANSCRIPTOMIC SINGLE CELL cDNA For the scRNAseq, Neocortices of P90 Emx/tdTom (control, n=4) and Emx/Tom/DTA (ablated, n=2) were microdissected and the tissue dissociated into single-cell suspensions, as previously described. Briefly, mice were transcardially perfused with ice-cold oxygenated artificial cerebrospinal fluid (22 mM NaCl, 0.63 mM KCl, 0.4 mM NaH2PO4 * 2H2O, 6.5 mM NaHCO3, 25 mM Saccharose, 5 mM Glucose, 0.5 mM CaCl2, 4 mM MgSO4. pH 7.3) and the brains collected. The tissue was sectioned at the vibratome in ice-cold artificial cerebrospinal fluid and the motor cortex and cingulate cortex were microdissected. Tissue dissociation was performed with the Adult Brain Dissociation Kit (Miltenyi Biotec) following manufacturer'sinstructions(red blood cells removal step was not included). After debris removal, the cells were resuspended in ice-cold 1% BSA in artificial cerebrospinal fluid, filtered with 30 μm filter (Sysmex Partec) and FACS sorted with the BD Influx System (USB. BD FACS™) to separate GFP+ and TdTom+OL lineage cells. A scRNA-Seq experiment set with a cohort of control and experimental mice was discarded due to strong batch effects, most likely due to stress during the FACS procedure For the snRNAseq, to assess the impact of dOPC ablation immediately when ablation occurs, cortical tissue was analysed unsing single-nucleus RNA sequencing (snRNA-seq) of antibody-hashed samples. Neonatal mice were sacrificed on the day of birth by decapitation, then the motor cortex region was dissected in icecold oxygenated cutting solution containing 87mM NaCl, 2.5mM KCl, 1.25mM NaH2PO4, 26mM NaHCO3, 0.5mM CaCl2, 4mM MgSO4, 75mM sucrose, and 20mM glucose, before samples were snapfrozen on dry ice in a sterile microtube. The samples were stored at -80°C until further processed. To isolate nuclei, tissue fragments from two animals with identical genotype were combined in nuclei isolation buffer (250mM sucrose, 25mM KCl, 5mM MgCl2, 10mM Tris buffer pH 8.0, 0.1% NP40, 0.1mM DTT and 0.4U/μl RNAse inhibitors), and gently homogenized with a plastic pestle on ice. Subsequently, the homogenate was filtered using a 40μm strainer, and the nuclear pellet was collected upon centrifugation. The pellet was then washed twice with Nuclei Wash and Resuspension buffer (NWR) containing 2% BSA in PBS and 0.4U/μl RNAse inhibitors. Finally, the nuclei were resuspended in ST Staining Buffer (ST-SB: 2% BSA, 0.02% Tween-20, 10mM Tris pH 7.5, 146mM NaCl, 1mM CaCl2, and 21mM MgCl2), filtered again with 40μm strainer and collected for inspection and counting. Hashtag antibody tagging was done as described previously with minor adjustments. Two sets of ablated and two sets of control nuclear samples (1M nuclei per 100μl of ST-SB) were first blocked with TruStain FcX™ (anti-mouse CD16/32, Biolegend #101319) before the addition of 1μg/100μl Mab414 nuclei hashing antibodies (TotalSeq™ A0451-54, Biolegend #682205, #682207, #682209, #682211). To remove the unbound antibodies, samples were washed twice with ST Wash Buffer (10mM Tris, 146mM NaCl, 1mM CaCl2, 21mM MgCl2, 2% BSA, 0.4U/μL of RNAse inhibitor) and again with NWR buffer to avoid the detergent and magnesium carry-over prior to sequencing For the scRNAseq, the sorted cells were processed with the Chromium Single Cell A Chip kit v2 and library prep with the Chromium Single Cell 3´Library & Gel Beads kit v2 (10X Genomics) according to the manufacturer's instructions. A total of 3,000 cells for each sample were loaded on the Chromium Single Cell A Chip, although a lower number of cells was recovered in singlet and passed the quality control. For the snRNAseq, the nuclei were resuspended in NWR, strained with a 20μm and 10μm strainer before counting, sample pooling and downstream processing with 10X platform for single-nuclei sequencing For the snRNAseq, 4 hastag antibodies were used to label our samples, from Biolegend TotalSeq™ A0451-54. A0451 (DTA) TTCCTGCCATTACTA, A0452 (DTA) CCGTACCTCATTGTT, A0453 (Ctrl) GGTAGATGTCCTCAG, A0454 (Ctrl) TGGTGTCATTCTTGA Illumina NovaSeq 6000 SRX23428312 GSM8042315_r1 SRP486498 PRJNA1070591 SRS20282772 GSM8042315 GSM8042315 RNA-Seq TRANSCRIPTOMIC SINGLE CELL cDNA For the scRNAseq, Neocortices of P90 Emx/tdTom (control, n=4) and Emx/Tom/DTA (ablated, n=2) were microdissected and the tissue dissociated into single-cell suspensions, as previously described. Briefly, mice were transcardially perfused with ice-cold oxygenated artificial cerebrospinal fluid (22 mM NaCl, 0.63 mM KCl, 0.4 mM NaH2PO4 * 2H2O, 6.5 mM NaHCO3, 25 mM Saccharose, 5 mM Glucose, 0.5 mM CaCl2, 4 mM MgSO4. pH 7.3) and the brains collected. The tissue was sectioned at the vibratome in ice-cold artificial cerebrospinal fluid and the motor cortex and cingulate cortex were microdissected. Tissue dissociation was performed with the Adult Brain Dissociation Kit (Miltenyi Biotec) following manufacturer'sinstructions(red blood cells removal step was not included). After debris removal, the cells were resuspended in ice-cold 1% BSA in artificial cerebrospinal fluid, filtered with 30 μm filter (Sysmex Partec) and FACS sorted with the BD Influx System (USB. BD FACS™) to separate GFP+ and TdTom+OL lineage cells. A scRNA-Seq experiment set with a cohort of control and experimental mice was discarded due to strong batch effects, most likely due to stress during the FACS procedure For the snRNAseq, to assess the impact of dOPC ablation immediately when ablation occurs, cortical tissue was analysed unsing single-nucleus RNA sequencing (snRNA-seq) of antibody-hashed samples. Neonatal mice were sacrificed on the day of birth by decapitation, then the motor cortex region was dissected in icecold oxygenated cutting solution containing 87mM NaCl, 2.5mM KCl, 1.25mM NaH2PO4, 26mM NaHCO3, 0.5mM CaCl2, 4mM MgSO4, 75mM sucrose, and 20mM glucose, before samples were snapfrozen on dry ice in a sterile microtube. The samples were stored at -80°C until further processed. To isolate nuclei, tissue fragments from two animals with identical genotype were combined in nuclei isolation buffer (250mM sucrose, 25mM KCl, 5mM MgCl2, 10mM Tris buffer pH 8.0, 0.1% NP40, 0.1mM DTT and 0.4U/μl RNAse inhibitors), and gently homogenized with a plastic pestle on ice. Subsequently, the homogenate was filtered using a 40μm strainer, and the nuclear pellet was collected upon centrifugation. The pellet was then washed twice with Nuclei Wash and Resuspension buffer (NWR) containing 2% BSA in PBS and 0.4U/μl RNAse inhibitors. Finally, the nuclei were resuspended in ST Staining Buffer (ST-SB: 2% BSA, 0.02% Tween-20, 10mM Tris pH 7.5, 146mM NaCl, 1mM CaCl2, and 21mM MgCl2), filtered again with 40μm strainer and collected for inspection and counting. Hashtag antibody tagging was done as described previously with minor adjustments. Two sets of ablated and two sets of control nuclear samples (1M nuclei per 100μl of ST-SB) were first blocked with TruStain FcX™ (anti-mouse CD16/32, Biolegend #101319) before the addition of 1μg/100μl Mab414 nuclei hashing antibodies (TotalSeq™ A0451-54, Biolegend #682205, #682207, #682209, #682211). To remove the unbound antibodies, samples were washed twice with ST Wash Buffer (10mM Tris, 146mM NaCl, 1mM CaCl2, 21mM MgCl2, 2% BSA, 0.4U/μL of RNAse inhibitor) and again with NWR buffer to avoid the detergent and magnesium carry-over prior to sequencing For the scRNAseq, the sorted cells were processed with the Chromium Single Cell A Chip kit v2 and library prep with the Chromium Single Cell 3´Library & Gel Beads kit v2 (10X Genomics) according to the manufacturer's instructions. A total of 3,000 cells for each sample were loaded on the Chromium Single Cell A Chip, although a lower number of cells was recovered in singlet and passed the quality control. For the snRNAseq, the nuclei were resuspended in NWR, strained with a 20μm and 10μm strainer before counting, sample pooling and downstream processing with 10X platform for single-nuclei sequencing For the snRNAseq, 4 hastag antibodies were used to label our samples, from Biolegend TotalSeq™ A0451-54. A0451 (DTA) TTCCTGCCATTACTA, A0452 (DTA) CCGTACCTCATTGTT, A0453 (Ctrl) GGTAGATGTCCTCAG, A0454 (Ctrl) TGGTGTCATTCTTGA Illumina NovaSeq 6000 SRX23428313 GSM8042316_r1 SRP486498 PRJNA1070591 SRS20282773 GSM8042316 GSM8042316 RNA-Seq TRANSCRIPTOMIC SINGLE CELL cDNA For the scRNAseq, Neocortices of P90 Emx/tdTom (control, n=4) and Emx/Tom/DTA (ablated, n=2) were microdissected and the tissue dissociated into single-cell suspensions, as previously described. Briefly, mice were transcardially perfused with ice-cold oxygenated artificial cerebrospinal fluid (22 mM NaCl, 0.63 mM KCl, 0.4 mM NaH2PO4 * 2H2O, 6.5 mM NaHCO3, 25 mM Saccharose, 5 mM Glucose, 0.5 mM CaCl2, 4 mM MgSO4. pH 7.3) and the brains collected. The tissue was sectioned at the vibratome in ice-cold artificial cerebrospinal fluid and the motor cortex and cingulate cortex were microdissected. Tissue dissociation was performed with the Adult Brain Dissociation Kit (Miltenyi Biotec) following manufacturer'sinstructions(red blood cells removal step was not included). After debris removal, the cells were resuspended in ice-cold 1% BSA in artificial cerebrospinal fluid, filtered with 30 μm filter (Sysmex Partec) and FACS sorted with the BD Influx System (USB. BD FACS™) to separate GFP+ and TdTom+OL lineage cells. A scRNA-Seq experiment set with a cohort of control and experimental mice was discarded due to strong batch effects, most likely due to stress during the FACS procedure For the snRNAseq, to assess the impact of dOPC ablation immediately when ablation occurs, cortical tissue was analysed unsing single-nucleus RNA sequencing (snRNA-seq) of antibody-hashed samples. Neonatal mice were sacrificed on the day of birth by decapitation, then the motor cortex region was dissected in icecold oxygenated cutting solution containing 87mM NaCl, 2.5mM KCl, 1.25mM NaH2PO4, 26mM NaHCO3, 0.5mM CaCl2, 4mM MgSO4, 75mM sucrose, and 20mM glucose, before samples were snapfrozen on dry ice in a sterile microtube. The samples were stored at -80°C until further processed. To isolate nuclei, tissue fragments from two animals with identical genotype were combined in nuclei isolation buffer (250mM sucrose, 25mM KCl, 5mM MgCl2, 10mM Tris buffer pH 8.0, 0.1% NP40, 0.1mM DTT and 0.4U/μl RNAse inhibitors), and gently homogenized with a plastic pestle on ice. Subsequently, the homogenate was filtered using a 40μm strainer, and the nuclear pellet was collected upon centrifugation. The pellet was then washed twice with Nuclei Wash and Resuspension buffer (NWR) containing 2% BSA in PBS and 0.4U/μl RNAse inhibitors. Finally, the nuclei were resuspended in ST Staining Buffer (ST-SB: 2% BSA, 0.02% Tween-20, 10mM Tris pH 7.5, 146mM NaCl, 1mM CaCl2, and 21mM MgCl2), filtered again with 40μm strainer and collected for inspection and counting. Hashtag antibody tagging was done as described previously with minor adjustments. Two sets of ablated and two sets of control nuclear samples (1M nuclei per 100μl of ST-SB) were first blocked with TruStain FcX™ (anti-mouse CD16/32, Biolegend #101319) before the addition of 1μg/100μl Mab414 nuclei hashing antibodies (TotalSeq™ A0451-54, Biolegend #682205, #682207, #682209, #682211). To remove the unbound antibodies, samples were washed twice with ST Wash Buffer (10mM Tris, 146mM NaCl, 1mM CaCl2, 21mM MgCl2, 2% BSA, 0.4U/μL of RNAse inhibitor) and again with NWR buffer to avoid the detergent and magnesium carry-over prior to sequencing For the scRNAseq, the sorted cells were processed with the Chromium Single Cell A Chip kit v2 and library prep with the Chromium Single Cell 3´Library & Gel Beads kit v2 (10X Genomics) according to the manufacturer's instructions. A total of 3,000 cells for each sample were loaded on the Chromium Single Cell A Chip, although a lower number of cells was recovered in singlet and passed the quality control. For the snRNAseq, the nuclei were resuspended in NWR, strained with a 20μm and 10μm strainer before counting, sample pooling and downstream processing with 10X platform for single-nuclei sequencing For the snRNAseq, 4 hastag antibodies were used to label our samples, from Biolegend TotalSeq™ A0451-54. A0451 (DTA) TTCCTGCCATTACTA, A0452 (DTA) CCGTACCTCATTGTT, A0453 (Ctrl) GGTAGATGTCCTCAG, A0454 (Ctrl) TGGTGTCATTCTTGA Illumina NovaSeq 6000 SRX23428314 GSM8042317_r1 SRP486498 PRJNA1070591 SRS20282774 GSM8042317 GSM8042317 RNA-Seq OTHER cDNA For the scRNAseq, Neocortices of P90 Emx/tdTom (control, n=4) and Emx/Tom/DTA (ablated, n=2) were microdissected and the tissue dissociated into single-cell suspensions, as previously described. Briefly, mice were transcardially perfused with ice-cold oxygenated artificial cerebrospinal fluid (22 mM NaCl, 0.63 mM KCl, 0.4 mM NaH2PO4 * 2H2O, 6.5 mM NaHCO3, 25 mM Saccharose, 5 mM Glucose, 0.5 mM CaCl2, 4 mM MgSO4. pH 7.3) and the brains collected. The tissue was sectioned at the vibratome in ice-cold artificial cerebrospinal fluid and the motor cortex and cingulate cortex were microdissected. Tissue dissociation was performed with the Adult Brain Dissociation Kit (Miltenyi Biotec) following manufacturer'sinstructions(red blood cells removal step was not included). After debris removal, the cells were resuspended in ice-cold 1% BSA in artificial cerebrospinal fluid, filtered with 30 μm filter (Sysmex Partec) and FACS sorted with the BD Influx System (USB. BD FACS™) to separate GFP+ and TdTom+OL lineage cells. A scRNA-Seq experiment set with a cohort of control and experimental mice was discarded due to strong batch effects, most likely due to stress during the FACS procedure For the snRNAseq, to assess the impact of dOPC ablation immediately when ablation occurs, cortical tissue was analysed unsing single-nucleus RNA sequencing (snRNA-seq) of antibody-hashed samples. Neonatal mice were sacrificed on the day of birth by decapitation, then the motor cortex region was dissected in icecold oxygenated cutting solution containing 87mM NaCl, 2.5mM KCl, 1.25mM NaH2PO4, 26mM NaHCO3, 0.5mM CaCl2, 4mM MgSO4, 75mM sucrose, and 20mM glucose, before samples were snapfrozen on dry ice in a sterile microtube. The samples were stored at -80°C until further processed. To isolate nuclei, tissue fragments from two animals with identical genotype were combined in nuclei isolation buffer (250mM sucrose, 25mM KCl, 5mM MgCl2, 10mM Tris buffer pH 8.0, 0.1% NP40, 0.1mM DTT and 0.4U/μl RNAse inhibitors), and gently homogenized with a plastic pestle on ice. Subsequently, the homogenate was filtered using a 40μm strainer, and the nuclear pellet was collected upon centrifugation. The pellet was then washed twice with Nuclei Wash and Resuspension buffer (NWR) containing 2% BSA in PBS and 0.4U/μl RNAse inhibitors. Finally, the nuclei were resuspended in ST Staining Buffer (ST-SB: 2% BSA, 0.02% Tween-20, 10mM Tris pH 7.5, 146mM NaCl, 1mM CaCl2, and 21mM MgCl2), filtered again with 40μm strainer and collected for inspection and counting. Hashtag antibody tagging was done as described previously with minor adjustments. Two sets of ablated and two sets of control nuclear samples (1M nuclei per 100μl of ST-SB) were first blocked with TruStain FcX™ (anti-mouse CD16/32, Biolegend #101319) before the addition of 1μg/100μl Mab414 nuclei hashing antibodies (TotalSeq™ A0451-54, Biolegend #682205, #682207, #682209, #682211). To remove the unbound antibodies, samples were washed twice with ST Wash Buffer (10mM Tris, 146mM NaCl, 1mM CaCl2, 21mM MgCl2, 2% BSA, 0.4U/μL of RNAse inhibitor) and again with NWR buffer to avoid the detergent and magnesium carry-over prior to sequencing For the scRNAseq, the sorted cells were processed with the Chromium Single Cell A Chip kit v2 and library prep with the Chromium Single Cell 3´Library & Gel Beads kit v2 (10X Genomics) according to the manufacturer's instructions. A total of 3,000 cells for each sample were loaded on the Chromium Single Cell A Chip, although a lower number of cells was recovered in singlet and passed the quality control. For the snRNAseq, the nuclei were resuspended in NWR, strained with a 20μm and 10μm strainer before counting, sample pooling and downstream processing with 10X platform for single-nuclei sequencing For the snRNAseq, 4 hastag antibodies were used to label our samples, from Biolegend TotalSeq™ A0451-54. A0451 (DTA) TTCCTGCCATTACTA, A0452 (DTA) CCGTACCTCATTGTT, A0453 (Ctrl) GGTAGATGTCCTCAG, A0454 (Ctrl) TGGTGTCATTCTTGA Illumina NovaSeq 6000 SRX23428315 GSM8042318_r1 SRP486498 PRJNA1070591 SRS20282775 GSM8042318 GSM8042318 RNA-Seq TRANSCRIPTOMIC SINGLE CELL cDNA For the scRNAseq, Neocortices of P90 Emx/tdTom (control, n=4) and Emx/Tom/DTA (ablated, n=2) were microdissected and the tissue dissociated into single-cell suspensions, as previously described. Briefly, mice were transcardially perfused with ice-cold oxygenated artificial cerebrospinal fluid (22 mM NaCl, 0.63 mM KCl, 0.4 mM NaH2PO4 * 2H2O, 6.5 mM NaHCO3, 25 mM Saccharose, 5 mM Glucose, 0.5 mM CaCl2, 4 mM MgSO4. pH 7.3) and the brains collected. The tissue was sectioned at the vibratome in ice-cold artificial cerebrospinal fluid and the motor cortex and cingulate cortex were microdissected. Tissue dissociation was performed with the Adult Brain Dissociation Kit (Miltenyi Biotec) following manufacturer'sinstructions(red blood cells removal step was not included). After debris removal, the cells were resuspended in ice-cold 1% BSA in artificial cerebrospinal fluid, filtered with 30 μm filter (Sysmex Partec) and FACS sorted with the BD Influx System (USB. BD FACS™) to separate GFP+ and TdTom+OL lineage cells. A scRNA-Seq experiment set with a cohort of control and experimental mice was discarded due to strong batch effects, most likely due to stress during the FACS procedure For the snRNAseq, to assess the impact of dOPC ablation immediately when ablation occurs, cortical tissue was analysed unsing single-nucleus RNA sequencing (snRNA-seq) of antibody-hashed samples. Neonatal mice were sacrificed on the day of birth by decapitation, then the motor cortex region was dissected in icecold oxygenated cutting solution containing 87mM NaCl, 2.5mM KCl, 1.25mM NaH2PO4, 26mM NaHCO3, 0.5mM CaCl2, 4mM MgSO4, 75mM sucrose, and 20mM glucose, before samples were snapfrozen on dry ice in a sterile microtube. The samples were stored at -80°C until further processed. To isolate nuclei, tissue fragments from two animals with identical genotype were combined in nuclei isolation buffer (250mM sucrose, 25mM KCl, 5mM MgCl2, 10mM Tris buffer pH 8.0, 0.1% NP40, 0.1mM DTT and 0.4U/μl RNAse inhibitors), and gently homogenized with a plastic pestle on ice. Subsequently, the homogenate was filtered using a 40μm strainer, and the nuclear pellet was collected upon centrifugation. The pellet was then washed twice with Nuclei Wash and Resuspension buffer (NWR) containing 2% BSA in PBS and 0.4U/μl RNAse inhibitors. Finally, the nuclei were resuspended in ST Staining Buffer (ST-SB: 2% BSA, 0.02% Tween-20, 10mM Tris pH 7.5, 146mM NaCl, 1mM CaCl2, and 21mM MgCl2), filtered again with 40μm strainer and collected for inspection and counting. Hashtag antibody tagging was done as described previously with minor adjustments. Two sets of ablated and two sets of control nuclear samples (1M nuclei per 100μl of ST-SB) were first blocked with TruStain FcX™ (anti-mouse CD16/32, Biolegend #101319) before the addition of 1μg/100μl Mab414 nuclei hashing antibodies (TotalSeq™ A0451-54, Biolegend #682205, #682207, #682209, #682211). To remove the unbound antibodies, samples were washed twice with ST Wash Buffer (10mM Tris, 146mM NaCl, 1mM CaCl2, 21mM MgCl2, 2% BSA, 0.4U/μL of RNAse inhibitor) and again with NWR buffer to avoid the detergent and magnesium carry-over prior to sequencing For the scRNAseq, the sorted cells were processed with the Chromium Single Cell A Chip kit v2 and library prep with the Chromium Single Cell 3´Library & Gel Beads kit v2 (10X Genomics) according to the manufacturer's instructions. A total of 3,000 cells for each sample were loaded on the Chromium Single Cell A Chip, although a lower number of cells was recovered in singlet and passed the quality control. For the snRNAseq, the nuclei were resuspended in NWR, strained with a 20μm and 10μm strainer before counting, sample pooling and downstream processing with 10X platform for single-nuclei sequencing For the snRNAseq, 4 hastag antibodies were used to label our samples, from Biolegend TotalSeq™ A0451-54. A0451 (DTA) TTCCTGCCATTACTA, A0452 (DTA) CCGTACCTCATTGTT, A0453 (Ctrl) GGTAGATGTCCTCAG, A0454 (Ctrl) TGGTGTCATTCTTGA Illumina NovaSeq 6000 SRX23428316 GSM8042319_r1 SRP486498 PRJNA1070591 SRS20282776 GSM8042319 GSM8042319 RNA-Seq OTHER cDNA For the scRNAseq, Neocortices of P90 Emx/tdTom (control, n=4) and Emx/Tom/DTA (ablated, n=2) were microdissected and the tissue dissociated into single-cell suspensions, as previously described. Briefly, mice were transcardially perfused with ice-cold oxygenated artificial cerebrospinal fluid (22 mM NaCl, 0.63 mM KCl, 0.4 mM NaH2PO4 * 2H2O, 6.5 mM NaHCO3, 25 mM Saccharose, 5 mM Glucose, 0.5 mM CaCl2, 4 mM MgSO4. pH 7.3) and the brains collected. The tissue was sectioned at the vibratome in ice-cold artificial cerebrospinal fluid and the motor cortex and cingulate cortex were microdissected. Tissue dissociation was performed with the Adult Brain Dissociation Kit (Miltenyi Biotec) following manufacturer'sinstructions(red blood cells removal step was not included). After debris removal, the cells were resuspended in ice-cold 1% BSA in artificial cerebrospinal fluid, filtered with 30 μm filter (Sysmex Partec) and FACS sorted with the BD Influx System (USB. BD FACS™) to separate GFP+ and TdTom+OL lineage cells. A scRNA-Seq experiment set with a cohort of control and experimental mice was discarded due to strong batch effects, most likely due to stress during the FACS procedure For the snRNAseq, to assess the impact of dOPC ablation immediately when ablation occurs, cortical tissue was analysed unsing single-nucleus RNA sequencing (snRNA-seq) of antibody-hashed samples. Neonatal mice were sacrificed on the day of birth by decapitation, then the motor cortex region was dissected in icecold oxygenated cutting solution containing 87mM NaCl, 2.5mM KCl, 1.25mM NaH2PO4, 26mM NaHCO3, 0.5mM CaCl2, 4mM MgSO4, 75mM sucrose, and 20mM glucose, before samples were snapfrozen on dry ice in a sterile microtube. The samples were stored at -80°C until further processed. To isolate nuclei, tissue fragments from two animals with identical genotype were combined in nuclei isolation buffer (250mM sucrose, 25mM KCl, 5mM MgCl2, 10mM Tris buffer pH 8.0, 0.1% NP40, 0.1mM DTT and 0.4U/μl RNAse inhibitors), and gently homogenized with a plastic pestle on ice. Subsequently, the homogenate was filtered using a 40μm strainer, and the nuclear pellet was collected upon centrifugation. The pellet was then washed twice with Nuclei Wash and Resuspension buffer (NWR) containing 2% BSA in PBS and 0.4U/μl RNAse inhibitors. Finally, the nuclei were resuspended in ST Staining Buffer (ST-SB: 2% BSA, 0.02% Tween-20, 10mM Tris pH 7.5, 146mM NaCl, 1mM CaCl2, and 21mM MgCl2), filtered again with 40μm strainer and collected for inspection and counting. Hashtag antibody tagging was done as described previously with minor adjustments. Two sets of ablated and two sets of control nuclear samples (1M nuclei per 100μl of ST-SB) were first blocked with TruStain FcX™ (anti-mouse CD16/32, Biolegend #101319) before the addition of 1μg/100μl Mab414 nuclei hashing antibodies (TotalSeq™ A0451-54, Biolegend #682205, #682207, #682209, #682211). To remove the unbound antibodies, samples were washed twice with ST Wash Buffer (10mM Tris, 146mM NaCl, 1mM CaCl2, 21mM MgCl2, 2% BSA, 0.4U/μL of RNAse inhibitor) and again with NWR buffer to avoid the detergent and magnesium carry-over prior to sequencing For the scRNAseq, the sorted cells were processed with the Chromium Single Cell A Chip kit v2 and library prep with the Chromium Single Cell 3´Library & Gel Beads kit v2 (10X Genomics) according to the manufacturer's instructions. A total of 3,000 cells for each sample were loaded on the Chromium Single Cell A Chip, although a lower number of cells was recovered in singlet and passed the quality control. For the snRNAseq, the nuclei were resuspended in NWR, strained with a 20μm and 10μm strainer before counting, sample pooling and downstream processing with 10X platform for single-nuclei sequencing For the snRNAseq, 4 hastag antibodies were used to label our samples, from Biolegend TotalSeq™ A0451-54. A0451 (DTA) TTCCTGCCATTACTA, A0452 (DTA) CCGTACCTCATTGTT, A0453 (Ctrl) GGTAGATGTCCTCAG, A0454 (Ctrl) TGGTGTCATTCTTGA Illumina NovaSeq 6000