title: Ion Torrent Proton sequencing; miRNA-seq data from healthy and DMD human cells, along skeletal muscle differentiation from induced pluripotent stem cells
design: miRNA-seq data from healthy and DMD human cells, along skeletal muscle differentiation from induced pluripotent stem cells
library_construction_protocol: Samples were collected using Qiazol. Human tissue-derived myotubes – Primary myoblasts were differentiated into myotubes. Cells were seeded at 600 cells/cm2 on 0.1 % gelatin coated cultureware in myoblast medium containing 1 mM Acid ascorbic 2P (A8960, Sigma-Aldrich). 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 B of the extraction protocol was used for miRseq. PartB RNA was quantified and purity/quality was assessed on the 2100 Agilent BioAnlayzer using the Agilent small RNA kit (5067-1548, Agilent). 10 ng of miRNA was reverse transcribed using the Ion Total RNA-seq kit v2 (4475936, Thermofisher Scientific) following the protocol of the manufacturer for small RNA libraries. The cDNA libraries were amplified and barcoded using Ion Total RNA-seq kit v2 and Ion Xpress RNA-seq Barcode Adapters 1-16 Kit (Thermofisher Scientific). The amplicons were quantified using Agilent High Sensitivity DNA kit before the samples were pooled in sets of fifteen.