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Sample GSM3445675 Query DataSets for GSM3445675
Status Public on Nov 20, 2018
Title InfectedVehicle_3
Sample type SRA
 
Source name Primary cells, HUVEC
Organisms Homo sapiens; Human gammaherpesvirus 8
Characteristics human cell type: HUVEC
virus infection: KSHV
rnase treatment: no
time point: 2 days post infection
Treatment protocol Some conditions included RNase R treatment of the purified RNA.
Growth protocol HUVEC cells were grown in EGM2 media.
Extracted molecule total RNA
Extraction protocol Total RNA was purified using miRNeasy System (Qiagen).
Total RNA samples were depleted of ribosomal RNA using RiboMinus Eukaryote System v2 (ThermoFisher). Ribo-depleted RNA was treated with 6 U of RNase R (Epicenter Biotechnologies) at 37° for 15 min. Libraries were generated using TruSeq Stranded Total RNA System (Illumina).
Paired-end 150bp RNA-Seq sequencing reads.
 
Library strategy RNA-Seq
Library source transcriptomic
Library selection cDNA
Instrument model Illumina HiSeq 4000
 
Description Ribominus RNA
Exp#5-R
Data processing Base calling was performed using RTA version 1.18.64.
Demultiplexing and fastq file generation were conducted using bcl2fastq.
RNA-seq reads were trimmed using Cutadapt (version 1.14) to remove sequencing adapters, prior to mapping to a custom h19+KSHV (NC_009333.1) reference genome with STAR (version 2.5.2b) in two-pass mode with parameters: first pass parameters (--outSAMstrandField None --outFilterMultimapNmax 20 --alignSJoverhangMin 8 --alignSJDBoverhangMin 1 --outFilterMismatchNmax 999 --outFilterMismatchNoverLmax 0.3 --alignIntronMin 20 --alignIntronMax 1000000 --alignMatesGapMax 1000000 --readFilesIn $r1 $r2 --readFilesCommand zcat --runThreadN 32 --outFileNamePrefix $sampleName --chimSegmentMin 20 --outSAMtype BAM Unsorted), second pass parameters (--outSAMstrandField None --outFilterType BySJout --outFilterMultimapNmax 20 --alignSJoverhangMin 8 --alignSJDBoverhangMin 1 --outFilterMismatchNmax 999 --outFilterMismatchNoverLmax 0.3 --alignIntronMin 20 --alignIntronMax 1000000 --alignMatesGapMax 1000000 --sjdbFileChrStartEnd pass1.uniq.sj.out.tab --chimSegmentMin 20 --outSAMtype BAM Unsorted)
For annotation, the GENCODE Human v19 reference gene annotation (GTF) combined with the KSHV gene annotation were used.
The chimeric alignments were detected using the –chimSegmentMin parameter by the STAR aligner. These chimeric alignments were used by CIRCexplorer version 1.1.1 for detecting circRNAs. circRNAs with at least 5 reads mapping to the head-to-tail splice junction in one sample were used for quantitative analyses.
For the identification of viral circular RNAs, total RNA treated or not treated with RNase R were aligned by STAR with two-pass alignment for accurate mapping to novel splicing junctions. The chimeric alignments were used to detect viral circRNAs with CIRCExplorer2.3.
Genome_build: hg19 (GRCh37) + KSHV (NC_009333.1)
Supplementary_files_format_and_content: bed files containing back-splice junctions from the second pass of STAR.
 
Submission date Oct 24, 2018
Last update date Nov 23, 2018
Contact name Joseph M Ziegelbauer
Organization name NIH / National Cancer Institute
Street address 10 Center Dr. 6N110
City Bethesda
State/province MD
ZIP/Postal code 20892
Country USA
 
Platform ID GPL25719
Series (1)
GSE121756 RNA sequencing for KSHV circular RNAs
Relations
BioSample SAMN10290101
SRA SRX4934316

Supplementary file Size Download File type/resource
GSM3445675_InfectedVehicle_3.back_spliced_junction.bed.gz 148 b (ftp)(http) BED
SRA Run SelectorHelp
Raw data are available in SRA
Processed data provided as supplementary file

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