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| Status |
Public on Nov 06, 2024 |
| Title |
O5 |
| Sample type |
SRA |
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| Source name |
Muscle
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| Organism |
Mus musculus |
| Characteristics |
tissue: Muscle
cell line: C2C12
cell type: myoblast
genotype: ChR2::tdTomato
treatment: Differentiation media for 6 days
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| Treatment protocol |
Suspended 100k cells in a 24 wellplate (n=6) for each genotype. Seeded the cells onto a 1mm thick fibrin hydrogel consisting of 8 mg of fibrinogen/mL of GM+ media. GM+ consists of high glucose DMEM with sodium pyruvate, l-glutamine and glucose. We add 10% fetal bovine serum, 1% l-glutamine and 1% pen-strep alongside 1 mg/mL of aminocaproic acid to form GM+. They were cultured in GM+ for two days prior to being swapped onto DM++ media. DM++ media consists of the same high glucose DMEM base but with 10% inactivated horse serum, 1% l-glutamine, 1% pen-strp, 1 mg/mL of aminocaproic acis and 0.05 uL/mL of IGF-1.
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| Growth protocol |
High glucose DMEM w/ l-glutamine, sodium pyruvate and glucose as the base, with 10% fetal bovine serum and 1% pen-strep for growth of these myoblasts.
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| Extracted molecule |
total RNA |
| Extraction protocol |
We extracted the multinucleated myofibril monolayers using the Rneasy mini kit with standard extraction protocols for adherent cell monolayers. Lysed the cells for 5 minutes and passed the lysate through the QIAshredder before continuing the Rneasy mini kit extraction protocol.
Ten microliters of undiluted total RNA was incubated with 10ul of NEB Dynabeads for polyaA+ RNA selection, eluted, and fragmented in 20ul. Illumina libraries were constructed using NEB Ultra II protocol; 5ul of the fragmented solution using half the amount of reagent and 1,2 PrD instead of enhancer in the ligation step, with 14 PCR cycles of final amplification and TS Singular index barcoding.Library sizes were quantified and verified by QPCR and on a Fragmen t Analyzer before loading for sequencing on a Singular G4 instrument in a 50-base paired-end configuration. Low-input samples were prepared by ribodepletion using the ZapR v2 protocol and mammalian-specific R-probe V2 kit at full volume (7.5 min fragmentation time).
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 4000 |
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| Description |
instrument model: Singular Genomics G4
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| Data processing |
Fastq files were generated with the Illumina off-line basecaller (olb) v. 1.9.4.
Reads were aligned against mm10 (Feb., 2009) using bwa mem v. 0.7.12-r1039 with flags –t 16 –f, and mapping rates, fraction of multiply-mapping reads, number of unique 20-mers at the 5´ end of the reads, insert size distributions and fraction of ribosomal RNAs were calculated using bedtools v. 2.25.0. In addition, each resulting bam file was randomly down-sampled to a million reads, which were aligned against hg19, and read density across genomic features were estimated for RNA-Seq-specific quality control metrics
Fastq files were processed using the nf-core/rnaseq v 3.11.1 pipeline [https://zenodo.org/records/10171269] in the Nextflow 22.10.4 environment, using the GRCm39 reference genome and ENSEMBL GRCm39.110 murine annotation.
Gene-level expression tables of counts and transcript-per-million (TPM) generated by STAR/Salmon processing were retrieved [Dobin2013, Soneson2015, Patro2017] .
Assembly: GRCm39 / ENSEMBL 110 annotation
Supplementary files format and content: Gene-level raw counts and transcript per million (TPM) data generated by Salmon, tab-separated format
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| Submission date |
Nov 06, 2024 |
| Last update date |
Nov 06, 2024 |
| Contact name |
Vincent Butty |
| E-mail(s) |
[email protected]
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| Organization name |
Massachusetts Institute of Technology
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| Department |
Biology / Koch Institute / Bioengineering / CEHS
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| Lab |
BioMicro Center / IGE
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| Street address |
77 Massachusetts Avenue Bldg 68-317A
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| City |
Cambridge |
| State/province |
MA |
| ZIP/Postal code |
02139 |
| Country |
USA |
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| Platform ID |
GPL21103 |
| Series (2) |
| GSE281241 |
Decoupling the Biochemical and Mechanical Effects of Muscle Exercise on Motor Neurons with 2.5D Actuating Matrices [240614Ram_RNA-seq] |
| GSE281242 |
Decoupling the Biochemical and Mechanical Effects of Muscle Exercise on Motor Neurons with 2.5D Actuating Matrices |
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| Relations |
| BioSample |
SAMN44607382 |
| SRA |
SRX26632909 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
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