|
| Status |
Public on Oct 01, 2017 |
| Title |
HSUR 2-positive sample IP 2 |
| Sample type |
SRA |
| |
|
| Source name |
Herpesvirus saimiri-transformed T cells
|
| Organism |
Callithrix jacchus |
| Characteristics |
cell line: Cj319 T
treatment: Transformed with HVS strain A11
development stage: Adult
sample type: IP
|
| Treatment protocol |
Three independent samples of 60 million HVS-infected marmoset T cells each were transfected with GFP antisense oligonucleotide and three independent samples of of 60 million HVS-infected cells were transfected with HSUR 2 antisense oligonucleotide to deplete the levels of HSUR 2. After 24 hours, each sample was crosslinked with psoralen at 254 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 (MPG PureBioTech). Bound RNA was released from the beads with TRIzol. Inputs and HSUR 2 pull down samples were purified and crosslinks were reversed before proceeding directly to library preparation.
Illumina's TruSeq Stranded mRNA LT kit
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2500 |
| |
|
| Description |
polyA RNA crosslinked to viral HSUR 2 ncRNA
HSUR2_IP_vs_HSUR2_input.txt
HSUR2_IP_vs_Control_IP.txt
11581X20
|
| Data processing |
Reads were aligned to marmoset genomic sequence (calJac3), Saimiriine herpesvirus 2 (Genbank: X64346.1) and splice junction sequences using Novoalign (2.08.03), allowing up to 50 alignments. Splice junction sequences were created with USeq (v8.8.8) MakeTranscriptome using CalJac3 Ensembl build 80 and X64346.1 gene annotations.
The resulting alignment file was processed with the USeq SamTranscriptomeParser, which selects the appropriate alignment location for each read and discards repetitive or low quality alignments. SamTranscriptomeParser also converts splice junction alignment coordinates back to genomic space.
USeq DefinedRegionDifferentialSeq application was used to count the number of reads aligning to each gene.
DESeq (v1.4.5) was used to detect gene count differences between HSUR2_IP and Control_IP or HSUR2_IP and HSUR2_input
Genome_build: cj3 and X64346.1
Supplementary_files_format_and_content: tab-delimited files containing read counts, log2 fold changes and -10 * log10(adjusted p-value) for each gene.
|
| |
|
| Submission date |
Mar 10, 2016 |
| Last update date |
May 15, 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 |
GPL21580 |
| Series (1) |
| GSE79082 |
Identification of viral ncRNA-mRNA interactions in Herpesvirus saimiri-infected marmoset T cells |
|
| Relations |
| BioSample |
SAMN04545383 |
| SRA |
SRX1625704 |
