|
| Status |
Public on Apr 25, 2012 |
| Title |
Mock_control2 |
| Sample type |
SRA |
| |
|
| Source name |
HepG2 cells
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: HepG2
sirna: Scrambled
|
| Treatment protocol |
Scrambled control or siRNA directed against METTL3 (Sigma-Aldrich) were transfected into HepG2 cells by using Oligofectamine reagent (Invitrogen). Cells were re-transfected after 48 and 96 hours, and harvested on day 7 to allow more efficient METTL3 silencing.
|
| 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). RNA samples were poly(A) selected, and libraries were prepared using the Illumina TruSeq RNA sample preparation protocol (cat.# FC-122-1001).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 |
Basecalls performed using CASAVA version 1.8
RNA-seq reads were mapped to the human genome (build hg18) using TopHat 1.2.0 software
Differentially expressed genes: the number of reads mapped to each of the ensembl genes (release 54) was counted using the HTSeq python package, with the “union” overlap resolution mode, and –stranded=no. The R package DESeq v1.5.24 within the Bioconductor framework was used for differential expression analysis
Differentially expressed exons: non-overlapping exonic regions were defined using the “dexseq_prepare_annotation.py” script provided as part of the R DEXseq package. The number of reads falling in each of the defined exonic regions was counted using the DEXseq script “dexseq_count.py” with parameters -a=0 to include multi-reads mapped to different locations of the genome, and –stranded=no. Finally Differential expression analysis was done using the R package DEXSeq within the Bioconductor framework.
Differentially expressed introns: non-overlapping intronic regions which also don't overlap any exon on either strand were defined using an in-house script. The number of reads falling in each of the defined regions was counted using a modified version of the “dexseq_count.py” DEXseq script , and differential expression analysis was done using the DESeqBioconductor package
Differentially expressed isoforms: Ensembl gtf file of all human genes (hg18 release 54) was re-processed using Cuffcompare v1.0.3 in order to add the missing tss_id and p_id attributes according to the user guide. The resulting gtf annotation file created by Cuffcompare was used as input to Cuffdiff v1.0.3 tool together with the fragment alignment files. Both Cuffcompare and Cuffdiff are part of the Cufflinks package
Genome_build: hg18
Supplementary_files_format_and_content: Tab delimited txt files, providing differential genes/isoforms/exons/introns together with their normalized intensities, fold change (FC), p values (pval), and adjusted p values(adjp)
|
| |
|
| 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) |
| GSE37001 |
METTL3 KD in HepG2 cells |
| GSE37005 |
Topology of the human and mouse m6A RNA methylomes revealed by m6A-seq |
|
| Relations |
| SRA |
SRX136604 |
| BioSample |
SAMN00849842 |
