|
| 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 |
