|
| Status |
Public on Jun 14, 2019 |
| Title |
input_RNA_biorep2 |
| Sample type |
SRA |
| |
|
| Source name |
HEK293
|
| Organism |
Homo sapiens |
| Characteristics |
cell type: human embryonic kidney cell derived
rip antibody: anti-FLAG
strain: N/A
background: N/A
allele: Flag-Dnd1
|
| Treatment protocol |
cells were transfected with ~35 µg/plate FLAG-DND1 or untagged DND1 (as a negative control), grown for 24hrs, then harvested in PLB (polysome lysis buffer)
|
| Growth protocol |
DO-RIP-seq was performed as described in (Nicholson et al., 2017). Briefly, 5 - 15 cm2 plates of HEK293 cells were grown to 80-90% confluency
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA from IPs was radiolabeled and run on a 10% TBE-Urea gel. RNA fragments from ~25-75 nucleotides were excised and extracted from the gel.
cDNA libraries were prepared using the NEBNext Multiplexing Small RNA Library Prep Set for Illumina with 14-17 rounds of amplification, following the manufacturer’s protocol with the exception that the 5’ RNA adapter was replaced with a custom 5’ adapter compatible with UMI (Unique Molecular Identifier) tagging (Kivioja et al., 2011)
Libraries were sequenced using Illumina HiSeq 2500.
|
| |
|
| Library strategy |
RIP-Seq |
| Library source |
transcriptomic |
| Library selection |
other |
| Instrument model |
Illumina HiSeq 2500 |
| |
|
| Description |
DND1_DO-RIP-seq.xlsx
|
| Data processing |
Processing of data was performed as described in Nicholson et al. 2017
After removal of adapter and UMI sequences, reads were mapped to hg19 using TopHat2 (Version ##).
Binding sites were identified using the binning and normalization procedure of Nicholson et al. 2017
IPs were compared to input samples to identify sites enriched in DND1 IPs.
Reads with a low pass filter of >3 reads and >1.5 fold enrichment over input were considered DND1 binding sites.
hg19
Output of tab-delimited text files with total number of sites values for each target
|
| |
|
| Submission date |
Jun 13, 2019 |
| Last update date |
Jun 14, 2019 |
| Contact name |
Victor A Ruthig |
| E-mail(s) |
Victor.Ruthig@CUAnschutz.edu
|
| Organization name |
University of Colorado Anschutz Medical Campus
|
| Department |
Pediatrics, Section of Developmental Biology
|
| Street address |
12800 E 19th Ave
|
| City |
Aurora |
| State/province |
CO |
| ZIP/Postal code |
80045 |
| Country |
USA |
| |
|
| Platform ID |
GPL16791 |
| Series (1) |
| GSE132719 |
The RNA-Binding Protein DND1 Acts Sequentially as a Negative Regulator of Pluripotency and a Positive Regulator of Epigenetic Modifiers Required for Germ Cell Reprogramming |
|
| Relations |
| BioSample |
SAMN12055425 |
| SRA |
SRX6068286 |
