|
| Status |
Public on Aug 05, 2013 |
| Title |
MGC3 |
| Sample type |
SRA |
| |
|
| Source name |
mural granulosa cells
|
| Organism |
Homo sapiens |
| Characteristics |
cell type: mural granulosa cells
stimulation protocol: GnRH antagonist, rFSH, hCG
etiology of infertility: male factor
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted using the miRNeasy Mini Kit (Qiagen), according to the manufacturer’s instructions.
RNA population containing the poly(A) tail was enriched by Sera-Mag Oligo(dT) Magnetic Particles (Thermo Scientific) and library was prepared with mRNA Library Prep Master Mix Set 1 for Illumina (New England Biolabs) according to the manufacturer´s protocols with a few optimized modifications: a) all the purification steps were carried out with Agencourt AMPure XP (Beckman Coulter Inc.) beads, except for purification of fragmented RNA where RNeasy MinElute kit (Qiagen) was used; b) fragmentation incubation time was shortened to 2 minutes to gain longer fragments suitable for long sequencing runs; c) size selection by agarose gel electrophoresis was not performed, the average length of the fragment was 420 bp, including 300 bp of insert and 120 bp of adapter sequences; and d) the template amount for PCR was 15 ng and 10 cycles were carried out. The library preparation stage included annealing of unique adapters to individual samples for further sample indexing and pooling of all six poly(A) RNA samples. Adapters as well as PCR primers were from NEXTflex DNA Barcodes (Bioo Scientific Corporation). 13 pM of the sample pool was used for template hybridization, clustering and amplification (corresponds to 2.17 pM of individual sample).
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2000 |
| |
|
| Data processing |
Base calling and filtering was done using instrument software real time analysis (RTA v1.12.4.2) with default parameters.
Adapter sequences were removed with cutAdapt v0.9.3.
Bowtie v0.12.7 was used to map sequences to human genome 19 (Hg19) allowing no mismatches between the seed and target and reporting only the best aligned match. Aligned sequenced fragments were concatenated into transcripts using the TopHat v1.2.0 algorithm.
The BAM files from TopHat were converted to SAM format by SAMtools v.0.1.18. To get the number of reads per each gene the SAM files and the reference genome were used as input for HTSeq (http://www-huber.embl.de/users/anders/HTSeq), where intersection-nonempty mode was used.
Differential mRNA expression analysis was performed using the R/Bioconductor package EdgeR.
Genome_build: UCSC hg19
Supplementary_files_format_and_content: RNA counts per gene for each sample generated by HTSeq.
|
| |
|
| Submission date |
Apr 29, 2013 |
| Last update date |
May 15, 2019 |
| Contact name |
Agne Velthut-Meikas |
| Organization name |
Tallinn University of Technology
|
| Department |
Department of Chemistry and Biotechnology
|
| Street address |
Akadeemia tee 15
|
| City |
Tallinn |
| ZIP/Postal code |
12618 |
| Country |
Estonia |
| |
|
| Platform ID |
GPL11154 |
| Series (2) |
| GSE46490 |
High-throughput RNA sequencing of human preovulatory cumulus and mural granulosa cells (mRNA) |
| GSE46508 |
Small RNA-seq of human granulosa cells reveals miRNAs in FSHR and aromatase genes |
|
| Relations |
| BioSample |
SAMN02087552 |
| SRA |
SRX272871 |
