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| Status |
Public on Feb 10, 2016 |
| Title |
HEPG2 total RNA rearranged at 50c IP |
| Sample type |
SRA |
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| Source name |
HEPG2 cell line
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| Organism |
Homo sapiens |
| Characteristics |
cell line: HepG2
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| Treatment protocol |
NA
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| Growth protocol |
NA
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| Extracted molecule |
total 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.Competitive 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 rearrangement 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).
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| Library strategy |
OTHER |
| Library source |
transcriptomic |
| Library selection |
other |
| Instrument model |
Illumina HiSeq 2000 |
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| Data processing |
Library strategy: M1A-seq
Illumina Casava1.8.2 software used for basecalling.
Sequenced reads were trimmed for adaptor sequence, then mapped to mm10 or hg19 whole genome using tophat version 2.0.12 with parameters - -M -p 3 --no-novel-juncs -g 5
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.
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, mm10 for mouse
Supplementary_files_format_and_content: Peak lists include peak middle coordinates for each cell type
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| Submission date |
Jul 02, 2015 |
| Last update date |
May 15, 2019 |
| Contact name |
Sharon Moshitch-Moshkovitz |
| E-mail(s) |
[email protected]
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| Organization name |
Sheba Medical Center
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| Department |
Cancer Research Center
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| Street address |
Laboratory wing
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| City |
Tel-Hashomer |
| ZIP/Postal code |
52621 |
| Country |
Israel |
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| 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 |
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| Relations |
| BioSample |
SAMN03838494 |
| SRA |
SRX1080148 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
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