|
| Status |
Public on Oct 31, 2017 |
| Title |
KMSC-KSHV_BAC36-latent-ip-R3 |
| Sample type |
SRA |
| |
|
| Source name |
MSC Cells
|
| Organism |
Homo sapiens |
| Characteristics |
infection status: latent
cell line: MSC
antibody: SYSY
treatment: None
|
| Treatment protocol |
See samples section
|
| Growth protocol |
Cells were grown at 37C, 5% CO2 in their respective growth media
|
| Extracted molecule |
polyA RNA |
| Extraction protocol |
Total RNA was extracted with Trizol and mRNA was purified using polydT beads. m6A-IP was then performed using an anti-m6A antibody from Synaptic Systems. The immunoprecipitated RNA (IP) and input for each sample was used to generate libraries.
10-25ng of mRNA was used for library preparation using the Illumina TruSeq stranded mRNA kit according to manufacturer’s protocol with two modifications. First, the elute-frag-prime stage was done at 80°C for 2 min to allow annealing without causing fragmentation. RNA was reverse transcribed into first strand cDNA using reverse transcriptase and random primers. Then, the second strand cDNA was synthesized using DNA Polymerase I and RNase H. The cDNA fragments then went through an end repair process with the addition of a single ‘A’ base followed by ligation of the adapters. The products were then purified and enriched by PCR amplification for 10 cycles to generate the final RNA-Seq library. The second modification was done to adjust the bead-DNA ratio to preserve smaller fragments during the adapter ligation double beads clean up step. A beads:DNA ratio of 1:3 instead of 1:1 was used. cDNA libraries were quantified and pooled for cBot amplification and subsequent sequencing on an Illumina HiSeq 2000.
|
| |
|
| Library strategy |
OTHER |
| Library source |
transcriptomic |
| Library selection |
other |
| Instrument model |
Illumina HiSeq 2000 |
| |
|
| Data processing |
Library strategy: MeRIP-seq
Fastq file generated with CASAVA
m6A-seq reads were aligned to the corresponding genome (human / rat / KSHV_BAC16 / KSHV_BAC36) using Tophat 2.
m6A peaks called by exomePeak.
Differential m6A peaks was detected by the differential peak discovery algorithm bltest.
Motif analysis of m6A peaks was performed using the MEME package.
The expression of transcripts and isoforms were calculated using cufflinks based on the input samples.
The differential expression of transcripts and isoforms were calculated using cuffdiff, based on the input samples.
Genome_build: hg19, rn5
Supplementary_files_format_and_content: tab-delimited text files include RPKM values for each Sample ...
|
| |
|
| Submission date |
Aug 25, 2017 |
| Last update date |
May 15, 2019 |
| Contact name |
Hui Liu |
| E-mail(s) |
[email protected]
|
| Organization name |
China University of Mining and Technology
|
| Street address |
#1 Daxue Road
|
| City |
Xuzhou |
| State/province |
Jiangsu |
| ZIP/Postal code |
221116 |
| Country |
China |
| |
|
| Platform ID |
GPL11154 |
| Series (1) |
| GSE93676 |
Viral and Cellular N6-methyladenosine (m6A) Epitranscriptomes in KSHV Life Cycle |
|
| Relations |
| BioSample |
SAMN07561560 |
| SRA |
SRX3135224 |
