|
| Status |
Public on Feb 10, 2016 |
| Title |
HEPG2 mRNA untreated input replicate E |
| Sample type |
SRA |
| |
|
| Source name |
HEPG2 cell line
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: HepG2
|
| Treatment protocol |
in the heat shock experiments cells were incubated at 43 degrees celsius for 4 hours prior to harvesting.
|
| Growth protocol |
in the Glucose starvation experiments cells were grown on glucose free medium for 4 hours before harvest. In S.Pombe Cells were grown in either EMM-N medium for 4 hours or in malt mating medium for 5 hours
|
| Extracted molecule |
polyA RNA |
| Extraction protocol |
Total RNA of the indicated cell types was extracted using 5PRIME RNA purification kit. Where indicated mRNA enriched RNA was purified by oligo dT selection.
Fragmentation was carried out using Ambion fragmentation reagents in 70ºc.Competetive elution was carried out using free m1A nucleoside (330 microMolar m1A in 10 mM Tris pH 7.4 ,375 mM NaCl 0.1% NP-40.
Immunoprecipitation was done using MBL anti-m1A antibody D345-3 mAb.Dimmroth rearrangment was performed by heating the samples to either 50ºC or 60ºC for 1H under pH 10.4 conditions.
Libraries were prepared according to NEB's instructions accompanying the NEBNext® Ultra™ RNA Library Prep Kit for Illumina (Cat# 7530).
|
| |
|
| Library strategy |
OTHER |
| Library source |
transcriptomic |
| Library selection |
other |
| Instrument model |
Illumina HiSeq 2000 |
| |
|
| Data processing |
Library strategy: m1A-seq
Illumina Casava1.8.2 software used for basecalling.
Sequenced reads were trimmed for adaptor sequence, then mapped to hg19 whole genome or S.pombe genome version ASM294v2.29 or S.cerevisea genome version S288C. Aligment was done using tophat version 2.0.12 with parameters - -M -p 3 --no-novel-juncs -g 5 and the relevant gff transcriptome annotation
Enriched intervals were identified using MACS2,in IP over input. FC>=2, FDR<=0.05
Peak middle was determined for peaks that appeared in both replicates.if no replicates were avialable FC>=4 was taken.
For PARSseq samples reads were trimmed using cutadapt and aligned to the human transcriptome (knownCanonical transcripts from UCSC) using bowtie2 (--local).
Only uniquely aligned reads were used for downstream analysis
For each position in the transcriptome number of read start at each base were counted. For this step only reads with more than 5 nts matching the transcript at the start of the read were counted
PARS-scores were calculted for each position in the transcriptome as described in Wan et al. 2014.
Genome_build: hg19 for human ,S.pombe genome version ASM294v2.29 , S.cervisea genome version S288C
Supplementary_files_format_and_content: Peak lists in excel format include peak middle coordinates for each cell type
|
| |
|
| Submission date |
Dec 15, 2015 |
| Last update date |
May 15, 2019 |
| Contact name |
Sharon Moshitch-Moshkovitz |
| E-mail(s) |
[email protected]
|
| Organization name |
Sheba Medical Center
|
| Department |
Cancer Research Center
|
| Street address |
Laboratory wing
|
| City |
Tel-Hashomer |
| ZIP/Postal code |
52621 |
| Country |
Israel |
| |
|
| Platform ID |
GPL11154 |
| Series (2) |
| GSE70485 |
m1A marks translation initiation sites in human and mouse messenger RNA [Sequencing] |
| GSE76058 |
m1A marks translation initiation sites in human and mouse messenger RNA |
|
| Relations |
| BioSample |
SAMN04338136 |
| SRA |
SRX1482549 |
