|
| Status |
Public on Jul 09, 2015 |
| Title |
S56 |
| Sample type |
SRA |
| |
|
| Source name |
sperm
|
| Organism |
Homo sapiens |
| Characteristics |
phenotype group (birth outcome): Group II-iii
source site: Serono - Toronto
sequencing date: 2014-04-02
cell type: sperm
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Neat frozen (-80°C) semen samples from ejaculate obtained with permission of male partner from couples undergoing fertility treatment at two clinics - Serono(S) and Harvard(H) - were thawed then immediately subjected to purification through a 50% gradient of Puresperm (NidaCon International AB) in order to obtain pure sperm
Sperm RNA was isolated and purified using a modified RNeasy (Qiagen) protocol and finally the RNA quality assessed for DNA contamination as described in paper
A total of 2 ng of unfractionated input RNA along with the recommended concentration of ERCC RNA spike-in controls (Life Technologies) was reverse transcribed and the resulting cDNA amplified using SeqPlex RNA Amplification (Sigma-Aldrich Co.) allowing for the use of samples with low quantity of spermatozoa
Amplified primer dimers were removed prior to library construction using Agencourt AMPure XP (Beckman Coulter) and libraries were constructed with 50 ng of amplified cDNA using NEBNext Ultra DNA Library Prep Kit for Illumina (New England Biolabs) kit.
6bp inline Illumina index primers allowed for multiplexing of samples (Index in file name)
Paired-end sequencing (2x51 + 6Index) was performed using an Illumina HiSeq 2500 sequencer
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2500 |
| |
|
| Description |
sperm RNA
Phenotype key
Group: IVF treatment/birth outcome
Group I: TIC/achived LB within one spermatogenic cycle
Group II-i: IUI/TIC - delayed past first 90 day cycle
Group II-ii: ART preceded by unsuccessful IUI or TIC
Group II-iii: ART
Group III-i: Site 2 (Harvard) samples
Group III-ii: Patients never achieving LB/female factor observed
|
| Data processing |
Basecalling Illumina RTA 1.17.21.3
demultiplexing (Casava 1.8.2, Illumina)
Alignment to hg19+ERCC+18S/28S rRNA using Bowtie39 (v. 2.2.3.0) and splice junction mapper Tophat38 (v.2.0.12)
Total number of reads (primary) aligning within each sample to 278605 RNA elements - comprising all UCSC annotated elements plus 842 previously identified elements occuring uniquely in sperm - were summed using MultiBamCov (bedtools 2.17.0)
Coverage for each element was normalized by element length, and percentile rank within each sample for every element was calculated
Genome_build: hg19 + ERCC + 18S/28S rRNA
Supplementary_files_format_and_content: ClusterData_PercRank_GEOSubmission.xlsx contains for each sample, percentile rank (within sample) of element abundance for each of ~280K genomic elements.
|
| |
|
| Submission date |
Feb 05, 2015 |
| 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 |
GPL16791 |
| Series (1) |
| GSE65683 |
Sperm RNA: A window to idiopathic infertile couples |
|
| Relations |
| BioSample |
SAMN03330143 |
| SRA |
SRX867133 |
