|
| Status |
Public on Dec 09, 2013 |
| Title |
S2L-PS |
| Sample type |
SRA |
| |
|
| Source name |
Fertile mature sperm
|
| Organism |
Homo sapiens |
| Characteristics |
storage method: Liquefied storage
purification method: PureSperm cushion
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Semen samples was collected in liquefied fraction, or pelleted fraction, then treated with PureSperm gradient centrifugation through a 50% PureSperm cushion, or Somatic Cell Lysis Buffer (0.1% sodium dodecyl sulfate [SDS], 0.5% Triton X-100).
The RNA-Seq libraries for each sperm sample were prepared in two stages using the Nugen Ovation kit (Nugen Inc., San Carlos, CA) for cDNA synthesis and amplification plus the Nugen Encore system for library preparation. The cDNA samples were subject to single primer isothermal amplification (SPIA) prior to sequencing library preparation. In brief, 20 ng of total RNA was subject to reverse-transcription. The cDNA synthesis used oligo dT and random hexamer primers followed by isothermal amplification (SPIA). The amplified cDNA was then fragmented by covaris sonicator and the fragment ends were repaired. The Illumina compatible PE adaptors with inline barcodes were ligated onto the cDNA products followed by 15 cycles of PCR enrichment.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2000 |
| |
|
| Data processing |
Image analysis, base calling and FASTQ generation were performed using the genome analyzer pipeline software CASAVA (version 1.8.2).
Inline demultiplexing was performed using software fastq_multx (Aronesty 2011)
Sequencing reads were mapped to hg19 of the human reference genome (NCBI genome build 37.2) plus human ribosomal 5S, 18S and 28S sequences using Novoalign (Novocraft Technologies v.2.08, Selangor, Malaysia) paired-end base default parameters.
The relative abundance of each transcript was calculated using Genomatix software (www.genomatix.de) and presented as FPKM (fragments per kilobase exon per million fragments mapped).
Genome_build: hg19
Supplementary_files_format_and_content: tab-delimited text files include each read's mapping location on reference genome
|
| |
|
| Submission date |
Jan 17, 2013 |
| Last update date |
May 15, 2019 |
| Contact name |
Stephen Krawetz |
| E-mail(s) |
steve@compbio.med.wayne.edu
|
| Phone |
313-577-6770
|
| Organization name |
Wayne State University School of Medicine
|
| Department |
Center for Molecular Medicine and Genetics
|
| Lab |
C.S. Mott Center for Human Growth and Development
|
| Street address |
275 E. Hancock
|
| City |
Detroit |
| State/province |
MI |
| ZIP/Postal code |
48201 |
| Country |
USA |
| |
|
| Platform ID |
GPL11154 |
| Series (1) |
| GSE43586 |
Evaluation of the effectiveness of semen collection and sperm purification methods for spermatozoa transcript profiling |
|
| Relations |
| SRA |
SRX218245 |
| BioSample |
SAMN01889155 |
