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| Status |
Public on Mar 26, 2018 |
| Title |
RNA-seq NPF3 |
| Sample type |
SRA |
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| Source name |
NPF
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| Organism |
Homo sapiens |
| Characteristics |
patient: 3
gender: Male
tissue: Prostate
cell type: Normal Prostate Fibroblast
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| Treatment protocol |
Cells were cultured in standard media to approximately 80% confluency before being trypsinised, resuspended in media and then centifuged to create cell pellets. Cells were stored at -80°C until RNA extraction.
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| Growth protocol |
Prostate tissue was chopped into small pieces, approximately 2 mm3, and digested overnight at 37°C in RPMI containing 10% fetal calf serum (FCS), 25 mM HEPES, 100 U/mL penicillin, 100 mg/mL streptomycin, 0.5 mg/mL fungizone, 100 mg/mL gentamicin, 225 U/mL Collagenase Type I and 125 U/mL Hyaluronidase Type II (Sigma Aldrich) as previously described [PMID: 23558784]. Cell suspensions were seeded in RPMI containing 5% FCS, penicillin/streptomycin, 1 nM testosterone (Sigma Aldrich) and 10 ng/mL bFGF (Millipore), a medium that selects for fibroblasts compared to other prostatic cell types. Cells were grown at 37°C in a humidified incubator with 5% O2 and 5% CO2. The first passage of cells typically reached confluency within 2 weeks. The cells were used between passages 2-6.
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| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was extracted using the RNeasy kit (Qiagen) with on-column DNase I digestion. RNA concentration and integrity was assessed on an Agilent Bioanalyzer 2100 using the Eukaryote Total RNA Nano Chip (Agilent). RNA (500 ng) was depleted of ribosomal RNA using RiboZero (Illumina, CA, USA).
Library preparation was performed for each sample using the Illumina TruSeq Stranded Total RNA sample kit (Illumina, CA, USA) according to the manufacturer’s protocol. TruSeq directional paired-end sequencing with 75bp chemistry was performed with six samples multiplexed per lane.
RiboZero RNA-seq
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2500 |
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| Description |
NPF3
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| Data processing |
75bp paired-end reads for 4 pairs of patient-matched CAF and NPF cells were processed using Trim Galore (version 0.2.8) for adapter trimming, Tophat (version 2.0.10) [PMID: 23618408] for mapping reads and Cufflinks (version 2.0.10) [PMID: 20436464] to assemble transcripts, both guided by GENCODE annotations (version 19) [PMID: 22955987]. Uniquely mapped properly paired reads were unambiguously assigned to exons of GENCODE genes (version 19) and counted using ht-seqcount (version 0.5.4p3) [PMID: 25260700], reads mapping to known rRNA loci were discarded using samtools procedures [PMID: 19505943].
Genome_build: hg19
Supplementary_files_format_and_content: file of counts data generated from Htseq count
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| Submission date |
Aug 15, 2016 |
| Last update date |
May 15, 2019 |
| Contact name |
Ruth Pidsley |
| E-mail(s) |
r.pidsley@garvan.org.au
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| Organization name |
Garvan Institute of Medical Research
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| Street address |
384 Victoria Street
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| City |
Darlinghurst |
| State/province |
NSW |
| ZIP/Postal code |
2010 |
| Country |
Australia |
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| Platform ID |
GPL16791 |
| Series (2) |
| GSE85606 |
Cancer Associated Fibroblasts are defined by a core set of epigenome changes that contribute to the tumor phenotype [RNA-seq] |
| GSE86260 |
Cancer Associated Fibroblasts are defined by a core set of epigenome changes that contribute to the tumor phenotype |
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| Relations |
| BioSample |
SAMN05574747 |
| SRA |
SRX2023085 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
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