title: NextSeq 500 paired end sequencing; RNA-seq, ATAC-seq and DNA-methylation array data of human oligodendrocyte-like cells derived from transdifferentiated fibroblasts
design: RNA-seq, ATAC-seq and DNA-methylation array data of human oligodendrocyte-like cells derived from transdifferentiated fibroblasts
library_construction_protocol: FibA, B and C were acquired from ATCC (PCS-201-010, CRL-2097, CCL-153, respectively). Fibroblasts derived from one individual with PLP1 643C>T mutation were acquired from Coriell Institute (ID:GM09546) while fibroblasts derived from three male individuals with PLP1 duplication were kindly provided by Prof. Odile Boespflug-Tanguy, LEUKOFRANCE Biobank, Biochemistry Department, Kremlin Bicetre Hospital, APHP, Paris. Fibroblasts from PMD patients were compared with sex and age matched controls as described in Table S2. For the DNA methylation assay neonatal fibroblasts were acquired from ATCC (PCS-201-010) and Coriell Institute (GM00041, GM08680), fibroblasts from middle aged donors were kindly provided by Jürgen Winkler (UKERf-4L6) and three lines were generated in UKM (UKM-F4AC). Fibroblasts from old aged donors were provided from Jürgen Winkler (UKER-fG3G, UKERf-82A, UKERf-O3H). The study was approved by the local ethical committees in Münster, Erlangen, Milan and Paris (AZ 2018-040-f-S, AZ 259_17B and Banca INSpe, CPPAU788, CNIL1406552, AFSSAPS B90298-60). Fibroblasts were cultured in fibroblast medium (FM): Dulbecco's Modified Eagle Medium high glucose (Sigma) containing 10% FBS (Gibco), 1% nonessential amino acids (NEAA) (Sigma), 1% GlutaMAX (Gibco), and 1% penicillin/streptomycin solution (Sigma). All cell lines were negative for HIV, HCV, HBV and mycoplasma contamination. Fibroblasts were seeded at a density of 12x103 cells/cm2 on a Poly-L-lysine (Sigma) / mouse Laminin (Sigma) coated plate in FM. The next day cells were transduced with the SF-SON lentivirus in fresh FM and 16h later the medium was changed to Glial Induction Medium (GIM). GIM consisted of DMEM-F12 (Gibco), 1:200 N2 supplement (Gibco), 1:100 B27 supplement lacking vitamin A (Gibco) and 1% penicillin/streptomycin/glutamine (Sigma) supplemented with 0,5 μM SAG (Cayman Chemical), 10 ng/mL PDGF (Peprotech), 200 μM AA (Sigma) and 20 ng/mL T3 (Sigma). GIM was changed every other day and the concentration of T3 was increased to 60ng/ml at day 4. Puromycin (1μg/ml, Gibco) selection of the transduced cells took place the first 5 days. At day 9 the GIM was replaced by glial differentiation medium (GDM) consisting of DMEM-F12, 1:200 N2 supplement, 1:100 B27 supplement lacking vitamin A, 1% penicillin/streptomycin/glutamine enriched by 0,5 μM SAG, 60 ng/mL T3, 10 ng/mL NT3 (Peprotech), 10 ng/mL IGF-I (Peprotech), 200 μM AA and 10 ng/mL PDGF. PDGF was replaced by 100 μM dbcAMP (Sigma) at day 12. Medium change was performed every other day.  Cells of low passage (below 10) were used for all direct reprogramming experiments. Conversion efficiency is defined as the number of O4+ cells divided by the total number of DAPI+ cells at day 16. Yield is defined as the number of O4+ cells at day 16 divided by the number of plated fibroblasts at day 0. Starting with cells in lysis buffer, the amount of used DNA is regulated by the used cell number For the ATAC-seq protocol, the TD buffer and the TDE1 transposase from the Nextera Kit (Illumina) were used. The fragments size of the library was analyzed with a HighSensitivity Kit (Bioanalyzer). The rest of the protocol was performed as described in Buenrostro JD et. al. 2015 but used NEB Q5 HotStart HiFi PCR MasterMix.
attributes.tag: Experimental Factor: protocol
attributes.value: ATAC-seq
attributes.tag: Experimental Factor: experimental variable
attributes.value: directly converted human oligodendrocytes