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| Status |
Public on Jan 01, 2017 |
| Title |
IF biological rep2 |
| Sample type |
SRA |
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| Source name |
motor neurons supplying IF muscle
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| Organism |
Mus musculus |
| Characteristics |
age: p6
cell type: motor neuron
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| Treatment protocol |
Individual motor pools were labeled by injecting 0.1% CTB-Alexa 555 into individual muscles of p4 mice. At p6, the animals were anaesthetized on ice, then decapitated and eviscerated. In all instances possible, bench spaces and tools were subsequently cleaned with RNAzap (Life Technologies). Ventral laminectomies were quickly performed in cold PBS and the unfixed spinal cords were embedded longitudinally, ventral side down, in OCT and frozen at -80°. The muscles were then examined under epifluorescence for specificity, and spinal cords from non-specific injections were discarded. Prior to cryosectioning, RNAse free 1.0 PEN MembraneSlides (Zeiss) were UV crosslinked for 30 minutes at optimal power. The cryostat was cleaned with 95% EtOH. Frozen blocks were cryosectioned at 12 µm, with the angle of the block adjusted to section the spinal cord at a perpendicular cutting surface. The MembraneSlides were then placed in a slide box and frozen at -80° for laser capture microdissection. Laser capture microdissection was performed on the PALM Microbeam System (Zeiss). A solution of 300 µl lysis buffer with 2.1 µl BME was prepared for cell lysis. Cells from a set of slides corresponding to a single animal were preserved in 40 µl of the lysis buffer/BME mixture. The surface area captured in um2 was recorded for each set of slides. Each biological replicate required approximately 1 ng of RNA, corresponding to approximately 170000 um2 of laser captured surface area. For RNA purification, samples from individual animals were pooled to provide at least 250000 um2 of laser captured surface area per biological replicate.
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| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was extracted with the Absolutely RNA Nanoprep Kit (Agilent). cDNA was synthesized using the Ovation RNA-Seq System V2 kit (Nugen). cDNA libraries were constructed using the Nextera DNA Sample Prep Kit (Illumina) and sequenced on an Illumina HiSeq 2000 to a depth of 25-30 million single-end reads per sample.
cDNA libraries were constructed using the Nextera DNA Sample Prep Kit (Illumina) and sequenced on an Illumina HiSeq 2000 to a depth of 25-30 million single-end reads per sample.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2000 |
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| Data processing |
Illumina Casava1.7 software used for basecalling.
Sequenced reads were trimmed for adaptor sequence, and masked for low-complexity or low-quality sequence, then mapped to mm9 whole genome using bowtie v0.12.9 with parameters -X 20000 -k 2 -m 2 -q -p. Reads Per Kilobase of exon per Megabase of library size (RPKM) were calculated using a protocol from Mortazavi et al., Nat Methods, 2008.
Differenetial gene expression was calculated using the R statistical package DESeq.
Genome_build: mm9
Supplementary_files_format_and_content: tab-delimited text files include RPKM values and differenetial gene expression for the samples
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| Submission date |
Dec 10, 2016 |
| Last update date |
May 15, 2019 |
| Contact name |
Alana Mendelsohn |
| Organization name |
Columbia University
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| Lab |
Rui Costa
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| Street address |
3227 Broadway
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| City |
New York |
| State/province |
NY |
| ZIP/Postal code |
10027 |
| Country |
USA |
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| Platform ID |
GPL13112 |
| Series (1) |
| GSE91402 |
Molecular distinctions between motor neurons supplying Tibialis anterior (TA), Extensor digitorum longus (EDL), Peroneus longus (PL), Gastrocnemius (GS) and Intrinsic foot (IF) muscles [RNA-seq] |
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| Relations |
| BioSample |
SAMN06130398 |
| SRA |
SRX2413950 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
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