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| Status |
Public on Sep 11, 2024 |
| Title |
L6mito_input60ng_0.1x |
| Sample type |
SRA |
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| Source name |
L6
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| Organism |
Rattus norvegicus |
| Characteristics |
cell line: L6
cell type: myoblast
Sex: male
input: input60ng
ratios of_adapters: 0.1x
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| Extracted molecule |
total RNA |
| Extraction protocol |
Mitochondria were isolated from approximately 5x10^6 L6 myoblast cells with the MACS Mitochondria Isolation Kit (mouse) (cat# 130-094-532, Miltenyi Biotec). Following isolation, the mitochondrial fraction was resuspended in 50 μL ice-cold Storage Buffer (cat# 130-094-532, Miltenyi Biotec) and incubated with 312 μg RNase-A (cat# 19101, Qiagen) for 1 h at 37°C. Following incubation, RNase-A was degraded by the addition of 5 μL proteinase-K (stock concentration 600 mAU/mL) (cat# 19131, Qiagen) and inverting the tube briefly. The solution was centrifuged at 8,000g for 10-minutes at 4C. The supernatant was aspirated, and the mitochondria pellet was washed twice with 100 μL ice-cold Storage Buffer (cat#130-096-946, Miltenyi Biotec), and centrifuged at 13,000g for 2-minutes at 4C between washes. The mitochondria pellet was resuspended in 100 μL storage buffer (cat# 130-094-532, Miltenyi Boitec) and stored at -80°C until further analyses. For RNA extraction from isolated mitochondria, frozen samples were suspended in 5x volumes of TriReagent (cat# R2061, Integrated Sciences), thawed on ice and then centrifuged at 16,000xg for 2 min at 4°C to pellet the mitochondria. The mitochondria pellet was first sheared 20x through a fine pipette tip. Total RNA was extracted from isolated mitochondrua using Zymo Direct-Zol Micro Prep kit (#R2061, Integrated Sciences) with on-column DNase-I digest (#E1010, Integrated Sciences) as per the manufacturer's protocol. RNA was eluted in 12ul NFW.
Small RNA libraries were prepared using the NEBNext Multiplex Small RNA Library Prep Set (cat# #E7560S, New England Biolabs.) with modifications Briefly, the 3’ and 5’ adapters and reverse transcription primer were diluted 0.5x, 0.3x, or 0.1x and 20 end-point PCR cycles were conducted. Following PCR amplification, the cDNA library was cleaned using the Monarch PCR & DNA Cleanup Kit (5 μg) (cat# T1030L, New England Biolabs.) using the 7:1 ratio of binding buffer:sample as per the manufacturer’s instructions. Uniquely indexed libraries were placed into an equimolar pool and were run in separate lands of a 6% Novex TBE-polyacrylamide gel (cat# EC6265BOX, Thermo Fisher Scientific), with 5 μL Quick-Load pBR322 DNA-MspI Molecular Marker (cat#E7560S, New England Biolabs.) run in a separate lane. Bands corresponding to the 143 and 190bp molecular markers were manually excised and extracted from the gel as per the manufacturer's protocol (cat#E7560S, New England Biolabs.).
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| Library strategy |
miRNA-Seq |
| Library source |
transcriptomic |
| Library selection |
size fractionation |
| Instrument model |
Illumina MiSeq |
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| Description |
L6M-60-C_S11_L001_R1_001
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| Data processing |
A 1.3 pM loading pool was prepared with 20% Phi-X spike-in and was sequenced on a single-408 end 75 bp run. Sequenced reads underwent quality checks with FastQC before the adapter and reads 409 <20nt were trimmed. Following sequencing, read1 was trimmed using fastp (fastp 0.20.0) and then mapped using blast (2.9.0+) with switch -task blastn and word length set to 19nt to miRbase mouse mature miRNAs (mature.fa download march 12 2018, miRbase v22.1). Raw read counts for each mature miRNA generated using short perl script.
Assembly: miRbase v22.1
Supplementary files format and content: .csv file includes raw read counts for each miRNA for each sample
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| Submission date |
Sep 04, 2024 |
| Last update date |
Sep 11, 2024 |
| Contact name |
Adam James Trewin |
| E-mail(s) |
adam.trewin@unimelb.edu.au
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| Phone |
0477840280
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| Organization name |
University of Melbourne
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| Department |
Anatomy and Physiology
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| Street address |
Royal Pde
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| City |
Parkville |
| State/province |
Victoria |
| ZIP/Postal code |
3010 |
| Country |
Australia |
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| Platform ID |
GPL19052 |
| Series (1) |
| GSE276303 |
Purification of mitochondria from skeletal muscle tissue for transcriptomic analyses reveals localization of nuclear-encoded non-coding RNAs [L6 mito] |
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| Relations |
| BioSample |
SAMN43494880 |
| SRA |
SRX25966282 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
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