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| Status |
Public on Mar 29, 2018 |
| Title |
induced iSLK.219 cells input rep 2 |
| Sample type |
SRA |
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| Source name |
induced iSLK.219 cells input
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| Organism |
Homo sapiens |
| Characteristics |
cell line: iSLK.219
cell type: KSHV-positive renal carcinoma cell line
genotype/variation: containing doxycycline-inducible KSHV.219
passages: 8
induced with: doxycyclin for 5 days
rip antibody: none
molecule subtype: Total RNA
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| Treatment protocol |
2.5 mg total cellular RNA was prepared from iSLK.219 cells that were either uninduced (latent virus), or induced for five days with doxycycline to allow KSHV lytic replication. 2 separate biological replicates for the uninduced and induced samples were used per treatment condition.
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| Growth protocol |
iSLK.219 cells were cultured in DMEM containing 10% Fetal Bovine Serum and 1% Pen-Strep
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| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was isolated and DNAse treated as in the m6A RIP, except the RNA was first fragmented to lengths of ~100 nt prior to immunoprecipitation with anti-m6A antibody (Synaptic Systems).
Immunoprecipitated RNA fragments and comparable amounts of input were subjected to first-strand cDNA synthesis using the NEBNext Ultra RNA Library Prep Kit for Illumina (New England Biolabs). Sequencing was carried out on Illumina HiSeq2500 according to the manufacturer’s instructions, using 10 pM template per sample for cluster generation, TruSeq SR Cluster kit v3 (Illumina), TruSeq SBS Kit v3-HS (Illumina) and TruSeq Multiplex Sequencing primer kit (Illumina).
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| Library strategy |
RIP-Seq |
| Library source |
transcriptomic |
| Library selection |
other |
| Instrument model |
Illumina HiSeq 2500 |
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| Description |
processed data file: human_Union_induced_FC4_genes.txt
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| Data processing |
library strategy: MeRIP-seq
Reads were mapped to reference human and KSHV transcriptomes using Bowtie.
m6A peaks were called and analyzed using the model-based analysis of ChIP-seq (MACS) peak-calling algorithm.
Human and viral peaks were considered significant if their MACS-assigned fold change was greater than four (FC>4) and individual False Discovery Rate value less than 5% (FDR>5%). For reference, we have included a full list of FC>2 peaks in the virus.
The above data processing steps were performed as described previously: Dominissini D, Moshitch-Moshkovitz S, Salmon-Divon M, Amariglio N, Rechavi G. Transcriptome-wide mapping of N(6)-methyladenosine by m(6)A-seq based on immunocapturing and massively parallel sequencing. Nat Protoc. 2013 Jan;8(1):176–89.
Genome_build: Human: University of California, Santa Cruz (UCSC) Dec. 2013 (GRCh38/hg38) KSHV: University of California, Santa Cruz (UCSC) KSHV Feb 2012 Assembly
Supplementary_files_format_and_content: Processed data files contain the genomic coordinates and transcript names for m6A peaks that map back to the human or KSHV transcriptome. Each processed data file is derived from the corresponding input and m6A IP conditions (uninduced1, induced1, uninduced2, induced2). For each of these conditions, two outputs are generated: m6A peaks in human transcripts and m6A peaks in viral transcripts. An additional output, "Human Union Induced" and "Human Union Uninduced" files refer to the human m6A peaks that occur in both biological replicates of the indicated treatment condition. "Chr" refers to chromosome number for m6A peaks occuring on human transcripts. KSHV is a non-segmented virus, so all m6A peaks mapping to KSHV by default occur on "chr1." "Peak_start" and "Peak_end" indicate the coordinates of the start and end of the m6A peak within the reference transcriptome. For m6A peaks in human transcripts, the RefSeq ID and gene symbol is provided. For m6A peaks in viral transcripts, the parameters "gene_start" and "gene_end" refer to the annotated start and stop sites for the viral open reading frame (ORF) containing the m6A peak. In cases where a single m6A peak spans more than one viral ORF, all of the annotated ORFs contained within that peak are listed. "Strand" indicates whether the indicated viral transcript is encoded on the positive or negative sense strand of the virus. "dist_middle_TSS" refers to the distance between the middle of the indicated m6A peak and the start of its respective ORF. "dist_middle_end" refers to the distance from the middle of the peak to the end of the ORF. FC refers to the "Fold Change" (m6A IP/input) for each peak. While only peaks with a fold change of four or greater (FC>4) were considered significant, for reference, all peaks of fold change two or greater (FC>2) are listed for KSHV transcripts.
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| Submission date |
Oct 05, 2017 |
| Last update date |
May 15, 2019 |
| Contact name |
Britt Glaunsinger |
| Organization name |
UC Berkeley
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| Department |
Plant and Microbial Biology
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| Lab |
565 Li Ka Shing Center
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| Street address |
1951 Oxford Street
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| City |
Berkeley |
| State/province |
CA |
| ZIP/Postal code |
94720 |
| Country |
USA |
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| Platform ID |
GPL16791 |
| Series (1) |
| GSE104621 |
N6-methyladenosine modification in lytic viral gene expression during Kaposi's sarcoma-associated herpesvirus infection [MeRIP-seq] |
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| Relations |
| BioSample |
SAMN07740596 |
| SRA |
SRX3244825 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
| Processed data are available on Series record |
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