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Sample GSM1565484 Query DataSets for GSM1565484
Status Public on Mar 18, 2015
Title METTL3 KO polyA(+) icSHAPE DMSO Biological Replicate 1
Sample type SRA
Source name Embryonic Stem Cells
Organism Mus musculus
Characteristics cell type: ES cells
cell line: J1
gender: male
Treatment protocol v6.5 cells were collected and incubated with either DMSO (mock modified) or NAI-N3 (icSHAPE modified) at 100mM final concentration in 10% final DMSO for 20 minutes at 37C
Growth protocol mESCs were grown on gelatin plates with serum and LIF.
Extracted molecule total RNA
Extraction protocol Mock or icSHAPE modified cells were harvested using Trizol LS, total RNA purified using RNeasy columns and processed for icSHAPE library construction.
Total or polyA selected RNA was first reacted with DIBO-Biotin (Life Technologies) to CLICK biotin to icSHAPE modified transcripts. RNA was then fragmented with RNA Fragmentation Reagent (Ambion) to sizes ranging between 25-150 nts. Fragmented RNA was 3’end repaired with T4 PNK (NEB) and 3’ adaptors ligated with T4 RNL2tr (NEB). cDNA synthesis was achieved with SuperScriptIII (Life Technologies) and icSHAPE modified transcripts were captured with MyOne C1 Streptavidin beads (Life Technologies). Enriched icSHAPE modified cDNAs were circularized and amplified using quantitative PCR before deep sequencing.
Library strategy OTHER
Library source transcriptomic
Library selection other
Instrument model Illumina NextSeq 500
Description PolyA (+) RNA, mock modified (DMSO)
Data processing Library strategy: icSHAPE
Raw sequencing reads were removed of PCR duplicates based on the randomized sequencing the 5’ barcode. The 5’ and 3’ barcodes/adaptors were then trimmed and reads were mapped to the mouse transcriptome (Ensembl annotation build GRCm38.74) using Bowtie2. For reads that can be mapped to multiple locations of the transcriptome, we randomly distribute them to ten different locations. The first nucleotide of each sequencing read from the NAI-N3 libraries are identified as the base directly 3’ of the icSHAPE modification.
Genome_build: Ensembl GRCm38.74 (mouse)
Supplementary_files_format_and_content: bigWig : the bigWig file provided contains processed data from the icSHAPE experiment. Data presented nucleotide reactivity at each base and is an intersection of the two biological replicates
Submission date Dec 15, 2014
Last update date May 15, 2019
Contact name Ryan Alexander Flynn
Organization name Stanford University
Street address 380 Roth Way, Room 265
City Stanford
State/province CA
ZIP/Postal code 94305
Country USA
Platform ID GPL19057
Series (2)
GSE60034 Dynamic landscape of RNA structures in vivo reveals principles of post-transcriptional regulation [icSHAPE]
GSE64169 Dynamic landscape of RNA structures in vivo reveals principles of post-transcriptional regulation
BioSample SAMN03265562
SRA SRX835939

Supplementary data files not provided
SRA Run SelectorHelp
Raw data are available in SRA
Processed data provided as supplementary file

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