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| Status |
Public on Jun 21, 2017 |
| Title |
189_outerCC |
| Sample type |
SRA |
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| Source name |
cumulus cells
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| Organism |
Bos taurus |
| Characteristics |
cell type: cumulus cells
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| Extracted molecule |
total RNA |
| Extraction protocol |
We extracted RNA from cumulus cells using the guanidinium thiocyanate-phenol chloroform procedure (Trizol reagent, Thermofisher), with the addition of 0.5ul GlycoBlue Coprecipitant (Thermofisher) as a RNA carrier. For the oocytes, we added 2l of the lysis buffer (20 IU/l RNase inhibitor (Amresco), 0.2% Triton X-100 (Amresco)) to the tube.
We eluted the RNA from CCs in 4ml of a solution containing dNTPs and oligo-dT30VN and proceeded with the polyA+ whole transcriptome amplification with the Smart-seq2 protocol for cDNA amplification. For the oocytes, we added 2l of the lysis buffer (20 IU/l RNase inhibitor (Amresco), 0.2% Triton X-100 (Amresco)) to the tube and proceeded with the polyA+ whole transcriptome amplification with the Smart-seq2 protocol for cDNA amplification. The samples were subjected to 16 cycles of PCR amplification. For all samples, we used one ng of amplified cDNA for library preparation using the Nextera XT DNA library preg Kit (Illumina, Inc), as and followed the procedures described in the Smart-seq2 protocol.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2500 |
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| Description |
RNA
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| Data processing |
The libraries were aligned against the Bos taurus genome UMD3.1 downloaded from Ensembl using the STAR (v2.5.2) aligner. Only reads with a unique match to the genome and less than five mismatches were further filtered to eliminate duplicates with picard (v2.5, http://broadinstitute.github.io/picard/).
The bam files containing non-duplicated reads were used as input for Cufflinks (v.2.2.1) together with Ensembl gene annotation UMD3.1.87 to obtain fragments per kilogram per million reads (FPKM) data. Genes were subjected to analytical procedures if FPKM>0.5 in eight or more cells of each group (oocytes, innerCC, outerCC).
The non-duplicated reads were submited to counting according to the Ensembl gene annotation UMD3.1.87 using HTSeq (v. 0.6.1) software.
Genome_build: UMD3.1
Supplementary_files_format_and_content: tab-delimited text files include FPKM values for all samples
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| Submission date |
Jun 05, 2017 |
| Last update date |
May 15, 2019 |
| Contact name |
Fernando H Biase |
| E-mail(s) |
[email protected]
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| Phone |
540-231-9520
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| Organization name |
Virginia Tech
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| Department |
School of Animal Sciences
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| Lab |
Biase lab
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| Street address |
175 West Campus Drive
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| City |
Blacksburg |
| State/province |
VA |
| ZIP/Postal code |
24061 |
| Country |
USA |
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| Platform ID |
GPL19172 |
| Series (1) |
| GSE99678 |
Functional signaling and gene regulatory networks between the oocyte and the surrounding cumulus cells |
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| Relations |
| BioSample |
SAMN07194163 |
| SRA |
SRX2883387 |
| 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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