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| Status |
Public on Oct 30, 2018 |
| Title |
037-B |
| Sample type |
SRA |
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| Source name |
KS skin tumor
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| Organism |
Human gammaherpesvirus 8 |
| Characteristics |
strain: KSHV (HHV-8)
patient diagnosis: Kaposi's sarcoma
tissue: skin biopsy
genotype: wild type (Uganda-C)
host gender: M
morphotype: Macular
hiv viral loads (log): 4.74
cd4 blood counts: 574
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| Extracted molecule |
polyA RNA |
| Extraction protocol |
Total RNA was isolated using the RNeasy mini kit with DNAse treatment step following manufacturer instructions. Total RNA from the 6 tumor samples analyzed by stranded sequencing were extracted using the NucleoSpin RNA kit (Machery-Nagle, Bethlehem, PA, USA). RNA was further concentrated and purified using the RNA Clean and Concentrator kit (Zymo Research, Irvine, CA, USA).
Unstranded RNA-seq libraries were prepared from 300 ng of total RNA using the TruSeq RNA Sample Prep Kit v2. In addition four KS tumor libraries were prepared using the TruSeq Stranded mRNA Library Kit (Illumina) from 100 ng of total RNA. Library size distributions were validated using an Agilent 2200 TapeStation (Agilent Technologies, Santa Clara, CA, USA). Additional library QC, pooling of indexed libraries, and cluster optimization was performed using Life Technologies’ Invitrogen Qubit® 2.0 Fluorometer (Life Technologies-Invitrogen, Carlsbad, CA, USA). The unstranded RNA-seq libraries were pooled (5-plex) and the stranded libraries were pooled (4-plex) and each pool was clustered onto a flow cell lane. Sequencing was performed using an Illumina HiSeq 2500 in “High Output” mode with a paired-end, 50 base reads (PE50) sequencing strategy for the unstranded libraries and non-paired end for the stranded libraries. Image analysis and base calling was performed using Illumina's Real Time Analysis v1.18 software, followed by 'demultiplexing' of indexed reads and generation of FASTQ files, using Illumina's bcl2fastq Conversion Software v1.8.2.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2500 |
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| Data processing |
Image analysis and base calling was performed using Illumina's Real Time Analysis v1.18 software, followed by 'demultiplexing' of indexed reads and generation of FASTQ files, using Illumina's bcl2fastq Conversion Software v1.8.4 (http://support.illumina.com/downloads /bcl2fastq_conversion_software_184.html).
Reads of low quality were filtered out prior to alignment to the KSHV (HHV-8) reference genome using TopHat v2.0.14. Counts were generated from TopHat alignments for each viral gene using the Python package HTSeq v0.6.1 ( http://www-huber.embl.de/users/anders/ HTSeq/doc/overview.html) using the KSHV NC_009333 UCDS ver 121715.gff, available on the series record.
Genome_build: NC_009333.1
Supplementary_files_format_and_content: tab-delimited text files include raw read count for feature in each sample; GFF file defines those features relative to NC_009333.1 genome
Supplementary_files_format_and_content: KSHV_reads_normalization_and_primary_transcript_calculations.xls: matrix table for all samples including raw read counts and counts normalized by the length of each feature in kilobases (RPK units) and additionally by sequencing depth (TPM units)
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| Submission date |
Jun 22, 2018 |
| Last update date |
Feb 07, 2019 |
| Contact name |
Timothy M Rose |
| E-mail(s) |
timothy.rose@seattlechildrens.org
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| Organization name |
Seattle Children’s Research Institute
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| Street address |
1900 Ninth Ave., 8th floor
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| City |
Seattle |
| State/province |
WA |
| ZIP/Postal code |
98101 |
| Country |
USA |
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| Platform ID |
GPL25233 |
| Series (1) |
| GSE116160 |
Quantitative RNAseq analysis of Ugandan KS tumors reveals KSHV gene expression dominated by transcription from the LTd downstream latency promoter |
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| Relations |
| BioSample |
SAMN09470211 |
| SRA |
SRX4283565 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM3211590_037_B_counts.txt.gz |
395 b |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
| Processed data are available on Series record |
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