organism: Homo sapiens
description: Protocols: Samples were collected using Qiazol. Human tissue-derived myoblasts - Primary myoblasts were maintained in a myoblast medium: DMEM/F-12, HEPES (31330-038, Thermo Fisher Scientific) supplemented with 10 % fetal bovine serum (FBS, Hyclone, Logan, UT), 10 ng/mL fibroblast growth factor 2 (FGF2, 100-18B, Peprotech), and 50 nM Dexamethasone (D4902, Sigma-Aldrich) on 0.1 % gelatin (G1393, Sigma-Aldrich) coated culture ware. Human induced pluripotent stem cells - Primary myoblasts were reprogrammed into hiPSCs following the protocol described in Massourides et al., using the Yamanaka's factors POU5F1, SOX2 and KLF4 transduction by ecotropic or amphotropic vectors (Table S3). HiPSCs were adapted and maintained with mTeSR™1 culture medium (05850, Stemcell Technologies) on Corning® Matrigel® Basement Membrane Matrix, lactose dehydrogenase elevating virus (LDEV)-Free-coated cultureware (354234, Corning Incorporated). Cells were then seeded at 20,000 cells/cm2, passaged and thawed each time with 10 μM StemMACS™ Y27632. Human iPSC-derived cell - Six hiPSCs (3 healthy and 3 DMD) were differentiated three times toward skeletal muscle lineage using commercial media designed from Caron's work (23) (Skeletal Muscle Induction medium SKM01, Myoblast Cell Culture Medium SKM02, Myotube Cell Culture Medium SKM03, AMSbio). This protocol is a 2D directed differentiation that uses 3 consecutive defined media (SKM01 from day 0 to 10, SKM02 from day 10 to 17 and SKM03 from day 17 to d25) and only one cell passage at day 10. RNA extraction was done using the miRNeasy Mini kit (217004, QIAgen) on the QIAcube instrument. RNAs coming from the part A of the extraction protocol was used for mRNA-seq. PartA RNA was quantified on Nanodrop spectrophotometer (ND-1000, Thermo Fisher Scientific) and purity/quality (RIN ≥ 7) was assessed on the 2200 TapeStation using the Agilent RNA ScreenTape (5067-5576 / 5067-5577 / 5067-5578, Agilent). Libraries are prepared with TruSeq Stranded mRNA kit protocol according supplier recommendations. Briefly, the key stages of this protocol are successively, the purification of PolyA containing mRNA molecules using poly-T oligo attached magnetic beads from 1µg total RNA, a fragmentation using divalent cations under elevated temperature to obtain approximately 300bp pieces, double strand cDNA synthesis and finally Illumina adapter ligation and cDNA library amplification by PCR for sequencing.
attributes.tag: ENA-FIRST-PUBLIC
attributes.value: 2020-03-01T04:03:08Z
attributes.tag: ENA-LAST-UPDATE
attributes.value: 2019-09-23T13:21:40Z
attributes.tag: External Id
attributes.value: SAMEA5790571
attributes.tag: INSDC center name
attributes.value: INSERM UEVE UMR861, I-STEM, AFM
attributes.tag: INSDC first public
attributes.value: 2020-03-01T04:03:08Z
attributes.tag: INSDC last update
attributes.value: 2019-09-23T13:21:40Z
attributes.tag: INSDC status
attributes.value: public
attributes.tag: Submitter Id
attributes.value: E-MTAB-8321:Tissue-derived DMD MyoT_03
attributes.tag: broker name
attributes.value: ArrayExpress
attributes.tag: cell line
attributes.value: M418
attributes.tag: cell type
attributes.value: Myotube
attributes.tag: common name
attributes.value: human
attributes.tag: developmental stage
attributes.value: Adult skeletal muscle
attributes.tag: disease
attributes.value: Duchenne muscular dystrophy
attributes.tag: genotype
attributes.value: Stop exon 7 c
attributes.tag: replicate
attributes.value: 1
attributes.tag: sample name
attributes.value: E-MTAB-8321:Tissue-derived DMD MyoT_03
attributes.tag: scientific_name
attributes.value: Homo sapiens