|
| Status |
Public on Apr 25, 2012 |
| Title |
Input1 |
| Sample type |
SRA |
| |
|
| Source name |
HepG2 cells
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: HepG2
antibody: none
|
| Growth protocol |
Cells were grown in DMEM (Gibco, Invitrogen Inc.) containing 4.5 g/L glucose and L-Glutamine supplemented with 10% FBS and penicillin/streptomycin.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was purified from the cells using 5PRIME PerfectPure RNA Cultured Cell kit (cat#2302340) and polyA+ selection was carried out using GenElute mRNA miniprep kit (Sigma Aldrich, cat#MRN70). RNA was fragmented chemically to ~100 nts long fragments and subjected to IP using anti-m6A antibodies. 1st strand cDNA was synthesized using Illumina random primers and Invitrogen SuperScript II Reverse Transcriptase (Cat# 18044-014) then 2strand cDNA was synthesized by Illumina's mRNA Sample preparation Kit (Part# 1004814). DNA was end-repaired using a combination of T4 DNA polymerase, E. coli DNA Pol I large fragment (Klenow polymerase) and T4 polynucleotide kinase. The blunt, phosphorylated ends were treated with Klenow fragment and dATP to yield a protruding 3- 'A' base for ligation of Illumina's adapters which have a single 'T' base overhang at the 3’ end. After adapter ligation ~200 bp DNA fragments (insert plus adaptor and PCR primer sequences) were isolated from 2% agarose gel. Purified DNA fragments were PCR amplified with Illumina primers for 15 cycles and 10pM DNA was captured on an Illumina flow cell for cluster generation. Libraries were sequenced on the Genome Analyzer IIx following the manufacturer's protocols.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina Genome Analyzer IIx |
| |
|
| Description |
human hepatocarcinoma cell line
|
| Data processing |
Supplementary_files_format_and_content: Supplementary data HepG2 untreated' file is provided in .txt format. It contains a dataset of all identified m6A peaks in HepG2 under the various treatments described above. Corresponding legends are: id – KnownCanonical identification; chr – chromosome; txStart – transcription start position on chromosome; txEnd – Transcription end position on chromosome; strand - +/- chromosome strand; geneSymbol – official symbol of gene; desc – description; Factor_iscoding– either coding or noncoding, depending on the annotation of the gene in the UCSC table; Meanexpquant – expression quantile of the gene based on reads in input experiment; Peaksnum – number of peaks identified in gene; Peakspos – position of peaks relative to beginning of transcript (after removing introns); Peaksexonnum – ordinal number of exons in which the peaks were identified; Peaksexlengths – lengths of the exons in which the peaks were found; Peakscores – the peakscore of the identified peaks; Annotpeakpos – segment along the gene in which the peaks.
|
| |
|
| Submission date |
Apr 03, 2012 |
| Last update date |
May 15, 2019 |
| Contact name |
Sharon Moshitch-Moshkovitz |
| E-mail(s) |
sharon.moshkovitz@sheba.health.gov.il
|
| Organization name |
Sheba Medical Center
|
| Department |
Cancer Research Center
|
| Street address |
Laboratory wing
|
| City |
Tel-Hashomer |
| ZIP/Postal code |
52621 |
| Country |
Israel |
| |
|
| Platform ID |
GPL10999 |
| Series (2) |
| GSE37003 |
m6A mapping in human RNA (untreated) |
| GSE37005 |
Topology of the human and mouse m6A RNA methylomes revealed by m6A-seq |
|
| Relations |
| SRA |
SRX136630 |
| BioSample |
SAMN00849869 |
