|
| Status |
Public on Oct 18, 2012 |
| Title |
FLAG-eIF4AIII:Y14 Rep 2 RIPiT-Seq |
| Sample type |
SRA |
| |
|
| Source name |
HEK293 cells
|
| Organism |
Homo sapiens |
| Characteristics |
cell type: Flp-In 293 T-Rex (Life technologies)
cell line: HEK293
antibody 1: anti-FLAG M2 (Sigma A2220)
antibody 1 manufactuer: Sigma
antibody 1 catalog #: A2220
antibody 2: anti-Y14 (4C4; Sigma Y1253)
antibody 2 manufactuer: Sigma
antibody 2 catalog #: Y1253
|
| Treatment protocol |
Expression of the FLAG-tagged protein was induced with tetracycline for ~16 hr. One hour prior to cell harvesting, cycloheximide (CHX) was added to 100 ug/ml. For formaldehyde crosslinking, cells were resuspended in PBS+CHX+0.1% formaldehyde. After gently mixing the suspension at room temperature for 10 minutes, reactions were quenched with one-tenth volume of quenching buffer (2.5 M Glycine, 25 mM Tris-base).
|
| Growth protocol |
Flp-In TRex-HEK293 cells containing a stable copy of FLAG-tagged EJC proteins routinely were grown in Dulbecco's modified eagle medium supplemented with 10% fetal bovine serum and 1% Penicillin-Streptomycin. For generating each EJC footprint library, cells were grown in three 15-cm plates for immunoprecipitation.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RIPiT-Seq: Total cell extracts generated by sonication in a buffer containing 300 mM NaCl were used for EJC immunoprecipition. RNA-Seq: PolyA+ RNA was extracted from Flp-In HEK293 cells and base-hydrolyzed. mRNP-Seq: Cleared nuclear extracts from Flp-In HEK293 cells were used to isolate footprints of polyA+ RNPs.
Size-selected RNA fragments were used to generate deep sequencing libraries using the small RNA expression kit (Applied Biosystems) following steps recommended by the manufacturer.
|
| |
|
| Library strategy |
OTHER |
| Library source |
transcriptomic |
| Library selection |
other |
| Instrument model |
AB SOLiD System 3.0 |
| |
|
| Description |
Short RNA footprint library from EJC tandem IP.
|
| Data processing |
Adapters were removed from short reads using ABI's SOLiD small RNA pipeline (version 0.5.0; custom-fitted with human genomic and exon junction reference sequences). Colorspace sequences were then converted to sequence space using Ma2Gff conversion tool (version 0.2.06). These gff files were converted to fasta files for further analysis and mapping. Before mapping, abundant non-coding RNAs (rRNAs, snoRNAs etc.) and repetitive sequences (SINEs, LINEs etc.) were filtered out. The rest of the reads were mapped to human genome and exon-junction database using Bowtie (0.12.7; parameters:-p 8 -v 1 -a --strata --best -m 1). The uniquely mapped reads are provided here as bed formats.
Genome_build: hg18
Supplementary_files_format_and_content: bed format alignments generated like in the step above.
|
| |
|
| Submission date |
Sep 25, 2012 |
| Last update date |
May 15, 2019 |
| Contact name |
Alper Kucukural |
| E-mail(s) |
alper.kucukural@umassmed.edu
|
| Phone |
508-856-2838
|
| Fax |
508-856-1002
|
| URL |
http://alper.kucukural.com
|
| Organization name |
UMass Medical School
|
| Department |
Biochemistry and Molecular Pharmacology
|
| Lab |
MooreLab
|
| Street address |
Lazare Medical Research Building, 870P
|
| City |
Worcester |
| State/province |
MA |
| ZIP/Postal code |
01605-4321 |
| Country |
USA |
| |
|
| Platform ID |
GPL9442 |
| Series (1) |
| GSE41154 |
The Cellular EJC Interactome Reveals Higher-Order mRNP Structure and an EJC-SR Protein Nexus |
|
| Relations |
| SRA |
SRX189572 |
| BioSample |
SAMN01730190 |
