|
| Status |
Public on Mar 18, 2015 |
| Title |
METTL3 KO polyA(+) icSHAPE DMSO Biological Replicate 2 |
| 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)
LIB_METTL3KO_polyA_icSHAPE_reactivity.bw
|
| 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 |
| E-mail(s) |
raflynn@stanford.edu
|
| 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 |
|
| Relations |
| BioSample |
SAMN03265531 |
| SRA |
SRX835940 |
