characteristics.text: ERC000011
characteristics.characteristic: ENA-CHECKLIST
characteristics.text: 2018-01-31T17:01:48Z
characteristics.characteristic: ENA-FIRST-PUBLIC
characteristics.text: 2017-08-08T13:38:40Z
characteristics.characteristic: ENA-LAST-UPDATE
characteristics.text: SAMEA104205894
characteristics.characteristic: External Id
characteristics.text: Departement de Cancerologie de l'Enfant et de l'Adolescent & UMR8203 \Vectorologie et Therapeutiques Anticancereuses\, CNRS, Gustave Roussy
characteristics.characteristic: INSDC center name
characteristics.text: 2018-01-31T17:01:48Z
characteristics.characteristic: INSDC first public
characteristics.text: 2017-08-08T13:38:40Z
characteristics.characteristic: INSDC last update
characteristics.text: public
characteristics.characteristic: INSDC status
characteristics.text: ERS1864912
characteristics.characteristic: SRA accession
characteristics.text: E-MTAB-5955:Q1
characteristics.characteristic: Submitter Id
characteristics.text: 6
characteristics.unit: week
characteristics.characteristic: age
characteristics.text: ArrayExpress
characteristics.ontologyTerms: http://www.ebi.ac.uk/efo/EFO_0009737
characteristics.characteristic: broker name
characteristics.text: skeletal muscle myoblast
characteristics.ontologyTerms: http://purl.obolibrary.org/obo/CL_0000515
characteristics.characteristic: cell type
characteristics.text: house mouse
characteristics.characteristic: common name
characteristics.text: Protocols: Freshly dissociated muscle cells were subjected to FACS sorting based on positive GFP-fluorescence and negative Propidium Iodide fluorescence purify quiescent muscle satellite cells. These isolated quiescent satellite cells were either collected for RNA extraction or seeded at 3,000 cells/cm2 in 1:1 DMEM:MCDB (Gibco and Sigma-Aldrich, respectively) containing 20% serum FBS (Gibco) and 1% Ultroser G (Pall) on Matrigel coated flasks (BD Biosciences) and cultured in an incubator under physiological oxygen pressure (37°C, 6.5% CO2, 3% O2). Sixty hours after plating, half the cells were gathered for RNA extraction and for the remaining cultures, medium was replaced to remove Ultroser G, and cells were further cultured for a total of 7 days to reach early differentiation. Six-weeks old male Tg:Pax7-nGFP mice were sacrificed by cervical dislocation, and their limb muscle were dissected, minced and digested in collagenase 0.1% and trypsin 0.25% at 37°C under gentle agitation. Cells were collected in serum-containing medium. For RNA collection, quiescent cells were directly sorted into Trizol-LS reagent (Invitrogen), and in-vitro cultured cells (activated at 60 hours and differentiated at 7 days) collected in Qiazol reagent (QIagen). Total RNA was subsequently purified using the miRNeasy Mini Kit following the manufacturer instructions (Qiagen). Ten micrograms of total RNA -obtained from several animals for the quiescent samples, were used for each biological replicate prepared for deep sequencing (i.e. 2 replicates for the quiescent and differentiated samples, and 3 replicates for the in vitro activated sample). For each biological replicate, 10 µg of total RNA (in 10 µl) were mixed with 10 µl of 2X TBE-Urea Sample Buffer (Invitrogen) and loaded in a well of a 15% polyacrylamide TBE-urea gel (Biorad). After migration, the gel was soaked in a SYBR gold (Invitrogen) solution, and imaged on a Dark Reader transilluminator. The 18-35 nucleotide region was cut using a scalpel for each sample, and the RNA eluted in 300 µl of 0.3 M NaCl solution under rotation for 4 hours at room temperature. The eluate was transferred together with gel debris onto a Spin X cellulose acetate filter (VWR) and centrifuged for 2 minutes at 12,000 xg. Small RNAs were finally precipitated by addition of 1 μl of glycogen (Invitrogen) and 750 μl of room temperature 100% ethanol followed by an incubation at -80°C for 30 minutes, and centrifugation for 25 minutes at 14,000 rpm and +4°C. The pellet was washed with 750 µl 75% Ethanol, dried and resuspended in 5 µl ultrapure water with 0.5 µl of RNAseOUT (Invitrogen). Small RNAs purified on gel were mixed to 1 µl of 10 µM pre-adenylated 3’ Illumina linker V1.5 (5'-rAppATCTCGTATGCCGTCTTCTGCTTG/3ddC/-3'), denatured for 2 minutes at 70°C, and further mixed with 1 µl of 10X T4 RNA-Ligase Truncated Reaction buffer, 0.8 µl 100 mM MgCl2, 0.5 µl RNaseOut and 1.5 µl of T4 RNA Ligase 2 truncated (New England Biolabs). Ligation was performed at 22°C for 1 hour. Then, 0.5µl of 5'-RNA adapter (5'-r(GUU CAG AGU UCU ACA GUC CGA CGA UC) -3'), 1 µl of 10 mM ATP and 1µl T4 RNA ligase (Ambion) were added, and ligation, performed at 20°C for 6 hours. Purified 5' and 3' ligated RNA in a volume of 4µl were then mixed with 1 µl of Solexa RT primer (20M) and denatured at 70°C and cooled on ice. Reverse transcription was then performed after addition of 2µl 5X first strand buffer (Invitrogen), 0.5µl of 12.5 mM dNTP mix, 1µl of 100 mM DTT, 0.5µl_ RNase OUT and 1 µl SuperScript III Reverse Transcriptase (Invitrogen) at 50°C for 1 hour, followed by 10 minutes at 70°C. The obtained cDNA was PCR amplified by addition of 27 µl Ultra-pure water, 10µl 5X Phusion-HF buffer, 1µl of 25µM Forward Primer (5’- AAT GAT ACG GCG ACC ACC GAC AGG TTC AGA GTT CTA CAG TCC GA -3’), 1µl of 25 µM revevrse Primer(5‘- CAA GCA GAA GAC GGC ATA CGA -3’), 0.5µl of 25 mM dNTP mix, and 0.5µl Phusion DNA Polymerase (Finnzymes) using 12 cycles 98°c 10 sec / 60°C 30 sec /72°C 15 sec. The library was finally purified on a 5% TBE PAGE gel, by cutting the region corresponding to the 92-106bp (the ligated linkers corresponding to a 73bp band visible on the gel). The gel was crushed by centrifugation and eluted in 1X Elution buffer (Illumina) by rotation for 2 hours at RT. The eluate was cleared using a Spin-X column and precipitated after addition of 1 µl of glycogen, 10 µl of 3M NaOAc and 325 µl of -20°C 100% ethanol, followed by a 20 min centrifugation at 14,000 rpm. After washing, the pellet was resuspended in 1ml dH2O. Finally, the sample was diluted to 10 nM and submitted to sequencing on a Solexa GA-IIX at the core sequencing facility.
characteristics.characteristic: description
characteristics.text: adult
characteristics.characteristic: developmental stage
characteristics.text: Tg:Pax7-nGFP
characteristics.characteristic: genotype
characteristics.text: Mus musculus
characteristics.characteristic: organism
characteristics.text: Mus musculus
characteristics.ontologyTerms: http://purl.obolibrary.org/obo/NCBITaxon_10090
characteristics.characteristic: organism
characteristics.text: musculature
characteristics.ontologyTerms: http://purl.obolibrary.org/obo/UBERON_0001015
characteristics.characteristic: organism part
characteristics.text: Quiescent
characteristics.ontologyTerms: http://purl.obolibrary.org/obo/PO_0020149
characteristics.characteristic: phenotype
characteristics.text: 1
characteristics.characteristic: replicate
characteristics.text: limb muscles and diaphragm
characteristics.characteristic: sampling site
characteristics.text: 0
characteristics.unit: day
characteristics.characteristic: sampling time point
characteristics.text: Mus musculus
characteristics.characteristic: scientific_name
characteristics.text: male
characteristics.ontologyTerms: http://purl.obolibrary.org/obo/PATO_0000384
characteristics.characteristic: sex
characteristics.text: B6D2
characteristics.characteristic: strain
characteristics.text: Q1
characteristics.characteristic: title