|
| Status |
Public on Jan 17, 2019 |
| Title |
oocyte_BCB_positive_1 |
| Sample type |
SRA |
| |
|
| Source name |
oocyte at geminal vesicle stage
|
| Organism |
Sus scrofa |
| Characteristics |
cell type: oocyte
cell developmental stage: germinal vesicle
bcb staining status: positive
|
| Treatment protocol |
BCB-negative and BCB-positive oocytes were respectively denuded off cumulus cells by gentle vortexing in 0.1% hyaluronidase. Following four washes using DPBS containing 1% BSA, the zona pellucida of denuded oocytes was removed using pronase (0.5% in DPBS). The zona pellucida-free oocytes were washed three times in DPBS/1% BSA and then each single oocyte with minimal amount wash buffer (<0.5µL) was placed into 4.35µL cell lysis buffer in a 0.2mL tube using pulled glass pipette. The same volume of DPBS with 1% BSA wash buffer added into lysis buffer was used as negative control to ensure the absence of contamination. The collected single oocyte samples were stored at -80℃.
|
| Growth protocol |
Pig ovaries were transported to the laboratory within 3 hours in a thermos bottle in physiological saline maintained at 30℃. Follicular fluid from 3- to 6-mm antral follicles were aspirated using an 18-gauge needle attached to a 10-ml disposable syringe. Cumulus-oocyte complexes (COCs) with at least three layers of intact cumulus cells and uniform ooplasm were selected for further procedure.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
The whole oocyte lysate of three single BCB-negative (numbered as 1, 2 and 3) and three single BCB-positive (6, 7 and 15) oocytes without maturation culture were used to amplify and prepare the sequencing library as previously described (Tang et al., 2010).
The first-strand cDNA was synthesized from the whole oocyte lysate using the UP1 primer containing a 24-nt poly(dT) tail at the 3′end, which allowed all RNAs with poly(A) tails to be reverse transcribed. Then, the first-strand cDNA were amplified by PCR using hot start EX Taq and UP2 primer containing 24-nt poly(dT) at the 3′end to add a universal tail to the second-strand cDNA. Amplified cDNA were purified from gel and evaluated using Agilent’s Bioanalyzer (Agilent Technologies). The prepared cDNAs were sheared into fragments to use for preparation of deep-sequencing libraries. All libraries were sequenced with an Illumina HiSeq 2000 instrument in a single lane.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina Genome Analyzer |
| |
|
| Description |
single cell RNA-seq
6
|
| Data processing |
Short reads were generated on the Illumina platform.
Clean reads were generated by removing low quality and adaptor sequence containing reads.
Clean reads were aligned to the Sscrofa10.2 genome assembly using BWA with default configurations.
After performing the assessment of sequence saturation and read distribution on reference genes, the expression levels of each gene were calculated as FPKM using the number of reads uniquely mapped to the specific gene and the total number of uniquely mapped reads in the sample using Cufflinks.
Genes and isoforms expression level are quantified by a software package: RSEM(RNASeq by Expectation Maximization).
Genome_build: Sscrofa10.2
Supplementary_files_format_and_content: text files containing gene and isoform expression levels.
|
| |
|
| Submission date |
Jul 11, 2017 |
| Last update date |
May 15, 2019 |
| Contact name |
Zhi-Qiang Du |
| E-mail(s) |
[email protected]
|
| Phone |
86-451-55191805
|
| Organization name |
Northeast Agricultural University
|
| Street address |
59 Mucai Street, XiangFang District
|
| City |
Harbin |
| ZIP/Postal code |
150030 |
| Country |
China |
| |
|
| Platform ID |
GPL9126 |
| Series (1) |
| GSE101181 |
Single Cell RNA-seq for porcine oocytes |
|
| Relations |
| BioSample |
SAMN07342406 |
| SRA |
SRX2995924 |
