|
| Status |
Public on Oct 08, 2017 |
| Title |
K8 CLIP-seq-1 in BCBL-1 cells |
| Sample type |
SRA |
| |
|
| Source name |
BCBL-1 cells
|
| Organisms |
Homo sapiens; Human gammaherpesvirus 8 |
| Characteristics |
infected with: Human gammaherpesvirus 8
clip antibody: K8 (generated and purified in our lab; Yan Wang, et al.,J Viral, 2011 and Xin Wang, et al., PLOS pathogens, 2015)
cell line: BCBL-1
cell type: primary effusion lymphoma cell line which carries latently infected KSHV
|
| Treatment protocol |
BCBL-1 was induced by 20ng/ml TPA for 48h
|
| Growth protocol |
BCBL-1,a primary effusion lymphoma cell line which carries latently infected KSHV and BJAB, a KSHV-free Burkitt lymphoma B cell line, were maintained in RPMI 1640 medium with 10% heat-inactivated fetal bovine serum (FBS) contained penicillin-streptomycin (50 U/ml), amphotericin B (1.25 μg /ml).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Turbo DNase was added into the cell lysate. Lysate was incubated on ice for 30 min and spun downed at 4°C and 20,000 g for 10 min to clear the lysate. Antibody of interest and 50μl washed protein A Dynabeads was added per 500μl lysates. Samples were rotated for 2 h at 4°C. Discarded the supernatant and washed the beads twice with 900 μl high-salt buffer. Then washed twice with 900 μl wash buffer with different mount of Rnase A (high-RNase concentration: 2μg/ml, moderate-RNase concentration: 1μg/ml and low-RNase concentration: 0.2μg/ml). Beads were washed with 900 μl RNase-free wash buffer twice. Then RNA 3’ ends were dephosphorylated. Linkers were ligated to 3’ ends of RNAs, 32P-γ-ATP labeled linker was ligated to the 5 ’ends of RNAs. The protein-RNA complexes were resolved on a 4-12% NuPAGE Bis-Tris gel and transferred to a nitrocellulose membrane.
K8-RNAs complexes were isolated and subjected to proteinase K digestion and RNA purification by TRIzol. RNAs were subjected to RT-PCR according to TruSeq™ RNA and DNA Sample Prep Kits.
|
| |
|
| Library strategy |
RIP-Seq |
| Library source |
transcriptomic |
| Library selection |
other |
| Instrument model |
Illumina HiSeq 2500 |
| |
|
| Data processing |
Sequence reads were trimmed for adaptor, then mapped to low-quality sequence hg19 and U75698.1 by TopHat 1.3.0 or bowtie software
Peak calling was performed by RIPSeeker software.
Genome_build: hg19 and U75698.1
Supplementary_files_format_and_content: txt file with peak coordinates.
|
| |
|
| Submission date |
Oct 06, 2017 |
| Last update date |
May 15, 2019 |
| Contact name |
Dongcheng Liu |
| E-mail(s) |
ldongch@mail2.sysu.edu.cn
|
| Organization name |
Sun Yat-sen University
|
| Department |
Institute of Human Virology and Ministry of Education Key Laboratory of Tropical Disease Control
|
| Street address |
zhongshan 2nd road
|
| City |
guangzhou |
| State/province |
guangdong |
| ZIP/Postal code |
510000 |
| Country |
China |
| |
|
| Platform ID |
GPL24095 |
| Series (1) |
| GSE104711 |
K8 CLIP-Seq in KSHV reactivated BCBL-1 cells |
|
| Relations |
| BioSample |
SAMN07757669 |
| SRA |
SRX3257823 |
