|
| Status |
Public on Sep 27, 2019 |
| Title |
control 2 |
| Sample type |
SRA |
| |
|
| Source name |
Herpesvirus saimiri-transformed T cells
|
| Organisms |
Callithrix jacchus; Saimiriine gammaherpesvirus 2 |
| Characteristics |
cell type: Cj319/cj137 T cell lines
transformation: Transformed with HVS strain A11
age: Adult marmoset
|
| Treatment protocol |
Three independent samples of 200 million HVS-infected marmoset T cells each were crosslinked with psoralen at 365 nm for one hour at 4°C. Samples were then lysed with 6 M guanidium hydrochloride, 0.8% SDS & proteinase K and incubated at 65°C for one hour. One milliliter of TRIzol was added to each sample and stored at -80°C.
|
| Growth protocol |
HVS-infected Marmoset T cells were grown at at density of 500,000-1 million cells/ml in RPMI 1640 medium supplemented with 20% FBS, 100 Units of penicllin, 100 µg/ml streptomycin, 1 mM of sodium pyruvate, 2 mM glutaMAX and 0.001% b-mercaptoethanol.
|
| Extracted molecule |
polyA RNA |
| Extraction protocol |
Total RNA was extracted with TRIzol following the manufacturer's protocol (Invitrogen). Poly A+ RNA was selected with oligo-dT magnetic beads following the manufacturer's protocol (New England Biolabs). Ten percent of each sample was saved as input and stored in TRIzol. Crosslinked-HSUR 2-mRNA complexes were captured with HSUR 2 antisense oligonucleotide and magnetic streptavidin beads (Dynabeads). Bound RNA was released from the beads with TRIzol. HSUR 2 and control pull down samples were purified and used directly to library preparation.
SMARTer Stranded Total RNA-Seq Kit v2 - Pico Input Mammalian (Takara)
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2500 |
| |
|
| Description |
polyA RNA crosslinked to viral HSUR 2 ncRNA
|
| Data processing |
A reference sequence file was created by combining the genomic sequences of Callithrix Jaccus (v3.2.1) and Saimiriine herpesvirus 2 (Genbank ID: X64346.1). A gene annotation file was created by combining Callithrix Jaccus Ensembl annotations (v91) and gene definitions from X64346.1. The STAR (v2.5.3a) ‘genomeGenerate’ command was used to create an index file using the reference and annotation files described above and with ‘sjdbOverhang’ set to 124.
Reads were processed using cutadapt (v1.6) to remove the first three base pairs at the start of the read and the ends of reads beginning at polyA stretches 10bp or longer. Reads shorter than 10bp after processing were removed from the analysis.
Trimmed reads were aligned to the reference index using STAR and set to report alignment data in bam format. The Subread ‘featureCount’ command was used to generate read counts for each gene in the annotation file described above, only counting reads on the same strand as the annotation.
DESeq2 (v1.14.1) was used normalize gene counts across the samples and determine differential expression between the enriched and non-enriched groups, controlling for sample.
Genome_build: CalJac3
Supplementary_files_format_and_content: tab-delimited text file containing EnsemblID, Gene Name, Log2 Fold Change, Adjusted P-value, and DESeq2 normalized counts.
Genome_build: X64346.1
|
| |
|
| Submission date |
Jan 18, 2019 |
| Last update date |
Sep 27, 2019 |
| Contact name |
Demian E Cazalla |
| E-mail(s) |
dcazalla@biochem.utah.edu
|
| Phone |
801 587 7466
|
| Organization name |
University of Utah
|
| Department |
Biochemistry
|
| Lab |
Demian Cazalla
|
| Street address |
15 N Medical Drive East, Rm 4100
|
| City |
Salt Lake City |
| State/province |
UT |
| ZIP/Postal code |
84112-5650 |
| Country |
USA |
| |
|
| Platform ID |
GPL26076 |
| Series (1) |
| GSE125371 |
Viral miRNA adaptor differentially recruits miRNAs to target mRNAs through alternative base-pairing |
|
| Relations |
| BioSample |
SAMN10777075 |
| SRA |
SRX5266920 |
