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| Status |
Public on Feb 25, 2024 |
| Title |
M3 |
| Sample type |
SRA |
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| Source name |
Nervous
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| Organism |
Mus musculus |
| Characteristics |
tissue: Nervous
cell line: HBG3 mESC HB9::GFP
cell type: Nerve cells
genotype: heterozygous blastocysts
treatment: Mechanical stimulation
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| Treatment protocol |
Once seeded onto our fibrin platform, myokine-infused medium was added to the spheroids or magnetic matrix actuation was applied for 30 mins at 4 Hz everyday throughout the length of the experiment.
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| Growth protocol |
HBG3 mESC were expanded on a feeder layer in mESC proliferation medium consisting of EmbryoMax ES DMEM with FBS, pen-strep, L-glut, EmbryoMax nucleosides, MEM non-essential amino acids, b-mercaptoethanol and mLIF. Then differentiated using a base of Advanced DMEM F-12 and Neurobasal with L-glutamine, pen-strep, KOSR, and b-mercaptoethanol. After moving into a low adhesion dish, puromorphamine and retinoic acid is added. After 5 days since initiating differentiation, we add ciliary neurotrophic factor and glial derived neurotrophic factor. Around day 8 of differentiation, the cells have aggregated into spheroids within the petri dish and have begun expressing HB9 signaling mature neuronal differentiation.
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| Extracted molecule |
total RNA |
| Extraction protocol |
RNeasy Micro Kit Protocol
Sequencing libraries were generated using Takara's SMARTR ZapR v2-based protocol; briefly, 4 ng of each RNA sample were used (corresponding to half of the reaction volume). Full volume was used for the 8 samples with the lowest concentration. RNAs were fragmented for 4 minutes, followed by first-strand cDNA synthesis using the SMARTR Pico v2 protocol. TruSeq-Singular unique dual index (UDI) sequencing adapters were added by PCR (5 cycles). Library fragments corresponding to rRNA molecules were depleted by cleaving with the ZapR v2 and mammalian-specific R-probe v2 kit at full volume, and the second round of PCR was performed using the Singular qPCR primers for 15 cycles. Library sizes were quantified and verified by QPCR and on a Fragment Analyzer before loading for sequencing on a Singular G4 instrument in a 50-base paired-end configuration.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2000 |
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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 |
Feb 25, 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 |
GPL13112 |
| Series (2) |
| GSE257540 |
Decoupling the Biochemical and Mechanical Effects of Muscle Exercise on Motor Neurons with 2.5D Actuating Matrices [230828Ram_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 |
SAMN40120782 |
| SRA |
SRX23732119 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
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