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| Status |
Public on Feb 19, 2016 |
| Title |
EX174179_V1N |
| Sample type |
SRA |
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| Source name |
Prostate cancer (Bone metastasis)
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| Organism |
Homo sapiens |
| Characteristics |
tissue: Bone
tumor stage: metastatic castration resistant
rin score: 2.4
median tin score: 51.72692313
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| Treatment protocol |
These 20 mCRPC patients were recruited as part of Prostate Cancer Medically Optimized Genome-Enhanced Therapy (PROMOTE) study. Tissue biopsies from a metastatic rib lesion were obtained before initiating enzalutamide treatment.
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| Extracted molecule |
polyA RNA |
| Extraction protocol |
Tissues were snap frozen with liquid nitrogen, and RNA was harvested using Rneasy Plus Mini Ki t(Qiagen). Ribo-zero rRNA remove Kit was used. Illumina TruSeq RNA Sample Prep Kit (Cat#FC-122-1001) was used
RNA libraries were prepared according to the manufacturer’s instructions for the TruSeq RNA Sample Prep Kit v2 (Illumina, San Diego, CA). The liquid handling Eppendorf (Hamburg, GER) EpMotion 5075 robot was employed for TruSeq library construction. All AMPure bead clean up, mRNA isolation, end repair and A-tailing reactions was completed on the 5075 robot. Reverse transcription and adaptor ligation steps were performed manually. Briefly, poly-A mRNA was purified from total RNA using oligo dT magnetic beads. The purified mRNA was fragmented at 95°C for 8 minutes, eluted from the beads and primed for first strand cDNA synthesis. The RNA fragments were then copied into first strand cDNA using SuperScript III reverse transcriptase and random primers (Invitrogen, Carlsbad, CA). Next, second strand cDNA synthesis was performed using DNA polymerase I and RNase H. The double-stranded cDNA was purified using a single AMPure XP bead (Agencourt, Danvers, MA) clean-up step. The cDNA ends were repaired and phosphorylated using Klenow, T4 polymerase, and T4 polynucleotide kinase followed by a single AMPure XP bead clean-up. The blunt-ended cDNAs were modified to include a single 3’ adenylate (A) residue using Klenow exo- (3’ to 5’ exo minus). Paired-end DNA adaptors (Illumina) with a single “T” base overhang at the 3’ end were immediately ligated to the ‘A tailed’ cDNA population. Unique indexes, included in the standard TruSeq Kits (12-Set A and 12-Set B) were incorporated at the adaptor ligation step for multiplex sample loading on the flow cells. The resulting constructs were purified by 2 consecutive AMPure XP bead clean-up steps. The adapter-modified DNA fragments were enriched by 12 cycles of PCR using primers included in the Illumina Sample Prep Kit. The concentration and size distribution of the libraries were determined on an Agilent Bioanalyzer DNA 1000 chip (Santa Clara, CA). A final quantification, using Qubit fluorometry (Invitrogen, Carlsbad, CA), was done to confirm sample concentration. mRNA libraries are loaded 2 per lane onto Illumina TruSeq v3 paired end flow cells at concentrations of 9 pM to generate cluster densities of 600,000-800,000/mm2 following Illumina’s standard protocol using the Illumina cBot and TruSeq Rapid Paired end cluster kit version 3.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2500 |
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| Description |
low RNA integrity group
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| Data processing |
Image data were processed using the Illumina Standard Pipeline.
Base-calling and data filtering processed by the Mayo Cliinc sequence core using the pipeline Cassava 1.7
Reads were aligned by TopHat 2.0.6 using the hg19 genome build and bowtie1 aligner option. Mean and standard deviation of fragment size were estimated by sampling the first 100,000 read pairs that were uniquely mapped.
Raw count, FPM and FPKM were calcualted using RSeQC (v2.3.6) with parameters "-d '1+-,1-+,2++,2--'
Genome_build: GRCh37 (hg19)
Supplementary_files_format_and_content: Processed files are Tab separated plain text files. Each file contains 9 columns: Chromosome, gene start coordinate, gene end coordinate, gene symbol, score size, strand, raw fragment count, FPM (fragment per million) and FPKM.
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| Submission date |
Jun 25, 2015 |
| Last update date |
May 15, 2019 |
| Contact name |
LIGUO WANG |
| E-mail(s) |
wang.liguo@mayo.edu
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| Organization name |
Mayo Clinic
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| Department |
Division of Computational Biology
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| Street address |
200 1st St SW
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| City |
Rochester |
| State/province |
MN |
| ZIP/Postal code |
55905 |
| Country |
USA |
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| Platform ID |
GPL16791 |
| Series (1) |
| GSE70285 |
Measure transcript integrity using RNA-seq data |
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| Relations |
| BioSample |
SAMN03795491 |
| SRA |
SRX1073702 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1722951_EX174179_V1N.FPKM.tsv.gz |
714.8 Kb |
(ftp)(http) |
TSV |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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