|
| Status |
Public on May 09, 2015 |
| Title |
FFPE_Enc_100_bc_tr |
| Sample type |
SRA |
| |
|
| Source name |
Sperm
|
| Organism |
Homo sapiens |
| Characteristics |
cell type: Fertile mature sperm
cdna amplification kit: Ovation RNA-Seq FFPE System (NuGEN)
rna-seq library preparation kit: Encore NGS Multiplex System I (NuGEN)
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Semen samples was treated with PureSperm gradient centrifugation through a 50% PureSperm cushion. RNA was isolated using the RNA isolation protocol as previously described [Goodrich et al. 2013]. In brief, a bead-based homogenization Qiazol protocol was applied to isolate the total RNA from each sample. An automated QiaCube extraction protocol specifically developed for sperm RNAs was used to extract the large RNA population. The residual DNA was removed by treating the isolated RNA with turbo-DNA Free (Ambion Inc.).
The RNA-Seq libraries for each sperm sample were prepared in two stages: cDNA amplification and RNA-Seq library preparation. In the first stage, four different amplification kits were studied: SMARTer™ Ultra Low RNA Kit for Illumina® Sequencing (Clontech Laboratories, Inc.), SeqPlex RNA Amplification Kit (Sigma-Aldrich Co.), Ovation® RNA-Seq System V2 and Ovation® RNA-Seq FFPE System from Nugen (NuGEN Technologies, Inc.). The RNA was reversed transcribed into cDNA then amplified. In the second stage, two library preparation kits were compared: Encore NGS Multiplex System I (NuGEN) and Ovation Ultralow Library Systems (NuGEN). The amplified cDNA is subject to sonication. Then the fragment ends are repaired before ligation of sequencing adaptors (with barcodes when necessary), followed by PCR amplification and library purification.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2500 |
| |
|
| Description |
Sample 4
|
| 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 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: bed files include each read's mapping location on reference genome
|
| |
|
| Submission date |
May 09, 2014 |
| 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) |
| GSE57503 |
Evaluation of RNA amplification and RNA-Seq library preparation protocols for spermatozoa RNA profiling |
|
| Relations |
| BioSample |
SAMN02768529 |
| SRA |
SRX539721 |
