GSM8187305: D14_Olig2; Mus musculus; RNA-Seq
For Ai14 reprogramming, LV-hGFAP::Sox10-P2A-Cre, LV-hGFAP::Olig2-P2A-Cre, LV-hGFAP::Nkx6.2-P2A-Cre, and control LV-hGFAP::BFP-T2A-Cre were used. For Ai14;Aldh1l1-CreERT2 reprogramming, LV-hGFAP::Sox10-P2A-zsGreen, LV-hGFAP::Olig2-P2A-zsGreen, LV-hGFAP::Nkx6.2-P2A-zsGreen and control LV-hGFAP::zsGreen were used. A multiplicity of infection (MOI) of 100 was used for all experiments. Virus-containing astrocyte media was placed on the cells and left overnight. Viral media was replaced with fresh astrocyte media one day post transduction (DPT). Three DPT, cells were switched to OPC differentiation media2 [DMEM/F12/Glutamax (Gibco Catalogue No.  11330032), 1X N2 (Gibco Catalogue No. 17502048), 1X B27 without vitamin A (Gibco Catalogue No. 17504044), 200 ng/ml SHH (R&D Systems Catalogue No.  464-SH), 20 ng/ml FGF (R&D Systems Catalogue No. 3139-FB), 4 ng/ml PDGF (Sigma Catalogue No. SRP3228)]. At 10 DPT, the cells were switched to OL differentiation media2 [DMEM/F12/Glutamax, 1X N2, 1X B27 without vitamin A, 40 ng/ml T3 (T2877 Catalogue No. Sigma), 200 ng/ml SHH, 100 ng/ml Noggin (R&D Systems Catalogue No. 1967-NG), 10 µM cAMP (Sigma Catalogue No. A9501), 100 ng/ml IGF (R&D Systems Catalogue No. 791-MG), 10 ng/ml NT3 (Sigma Catalogue No. SRP6007)]. At 14DPT, LV-hGFAP::Sox10-P2A-Cre, LV-hGFAP::Olig2-P2A-Cre, LV-hGFAP::Nkx6.2-P2A-Cre, and control LV-hGFAP::BFP-T2A-Cre cultures were processed using the BD Rhapsody System (BD Biosciences) and then sequenced. For single-cell isolation, an average of 9813.75 viable cells were captured in wells at cell load (Table S1). The BD Rhapsody scanner reported an average multiplet rate of 10.13% and an average number of wells with viable cells and a bead of 7081.5 (Table S1). Detailed metrics for each sample can be found in Table S1. Samples were down-sampled to 2500 cells and carried through and converted to cDNA using the BD Rhapsody WTA Reagent Kit (Becton Dickinson Canada, Catalogue No. 633802). Each cell was sequenced at approximately 100 million reads per cell (at least 2x150 bp paired-end reads) on a Novaseq (Donnelly Sequencing Centre, University of Toronto). In addition, LV-GFAP::Sox10 and control LV-GFAP::Cre cultures were collected prior to transduction, at 3DPT and 8DPT, and processed using the BD Rhapsody System (BD Biosciences) and then sequenced. For single-cell isolation, an average of 10263.8 viable cells were captured in wells at cell load (Table S2). The BD Rhapsody scanner reported an average multiplet rate of 6.86% and an average number of wells with viable cells and a bead of 7785 (Table S2). Detailed metrics for each sample can be found in Table S2. Samples were down-sampled to 2500 cells and carried through and converted to cDNA using the BD Rhapsody WTA Reagent Kit (Becton Dickinson Canada, Catalogue No. 633802). Each cell was sequenced at approximately 100 million reads per cell (at least 2x150 bp paired-end reads) on a Novaseq (Donnelly Sequencing Centre, University of Toronto). In addition, LV-GFAP::Sox10 and control LV-GFAP::Cre cultures were collected prior to transduction, at 3DPT and 8DPT, and processed using the BD Rhapsody System (BD Biosciences) and then sequenced. For single-cell isolation, an average of 10263.8 viable cells were captured in wells at cell load (Table S2). The BD Rhapsody scanner reported an average multiplet rate of 6.86% and an average number of wells with viable cells and a bead of 7785 (Table S2). Detailed metrics for each sample can be found in Table S2.