|
| Status |
Public on Aug 30, 2013 |
| Title |
E4_L2 and E4_L2HS |
| Sample type |
SRA |
| |
|
| Source name |
Flp-in T-REx 293-PTH-AGO1
|
| Organism |
Homo sapiens |
| Characteristics |
cell type: Flp-In T-REx 293
sample type: PTH-AGO1 bound RNAs
barcode: CACAGC
|
| Treatment protocol |
Cells were induced for PTH-AGO1 expression with Doxycycline for 36 hrs. Then existing protein-RNA complexes were stabilized by UV irradiation of living cells.
|
| Growth protocol |
Flp-In T-REx 293 cells were stably transfected with a plasmid encoding AGO1 N-terminally tagged with ProteinA-TEV protease cleavage site-Hisx6. Expression of tagged AGO1 is tetracycline inducible. Cells were grown in Dulbecco’s modified Eagle medium (DMEM) with high glucose, supplemented with 10% fetal bovine serum at 37°C under humidified air with 5% CO2.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
UV stabilized protein-RNA interactions were purified under denaturing conditions. RNAs associated with AGO1 were partially truncated, and the ends of RNA-duplexes formed within AGO1 RNA binding pocket were ligated together.
Sequencing 3' adapter was ligated while AGO1-RNA complexes were bound to Ni-NTA agarose, 5' adapter (barcoded for samples E2-E10) was ligated in solution after RNA isolation. RNA library was reverse transcribed and PCR amplified.
|
| |
|
| Library strategy |
OTHER |
| Library source |
transcriptomic |
| Library selection |
other |
| Instrument model |
Illumina Genome Analyzer IIx |
| |
|
| Data processing |
Demultiplexing: Illumina reads were assigned to experimental and control samples using their 5' barcode sequences, and barcodes were clipped
Collapsing reads: Identical sequencing reads were treated in the analysis as one, information on the actual numbers of reads is maintained in the sequence IDs: for E1-E4: X_Y and for E5-E6: X-Z_Y where X is frequency rank of the sequence, Y is a number of identical reads and Z corresponds to the number of random barcodes associated with identical reads
Reads alignment: Collapsed reads were aligned to a custom transcriptome database by BLAST, version 2.2.24; the components of the BLAST database come from BioMart, NCBI, genomic tRNA database and MirBase; more details in Helwak et. al. Cell 2013
Chimeras identification: We identified as chimeras those reads where: (1) the read yielded at least two blast hits with e-value < 0.1 against the human transcriptome database; (2) the hits were either directly adjacent in the read, or with up to 4 nucleotides gap or overlap between hits; and (3) the hits were in sense orientation with respect to the blast database. We rejected reads with more than ten hits in the database. A custom script was used for the purpose.
The energy of identified interactions was determined by scripts from UnaFold suite (by Markham and Zucker)
Supplementary_files_format_and_content: all processed data files are in the custom .hyb format
|
| |
|
| Submission date |
Aug 29, 2013 |
| Last update date |
May 15, 2019 |
| Contact name |
Grzegorz Kudla |
| E-mail(s) |
gkudla@gmail.com
|
| Phone |
+44 (0) 131 332 2471
|
| Organization name |
University of Edinburgh
|
| Department |
MRC HGU
|
| Street address |
Crewe Road
|
| City |
Edinburgh |
| ZIP/Postal code |
EH4 2XU |
| Country |
United Kingdom |
| |
|
| Platform ID |
GPL10999 |
| Series (1) |
| GSE50452 |
High-throughput identification of miRNA targets using CLASH (crosslinking, ligation and sequencing of hybrids) |
|
| Relations |
| BioSample |
SAMN02339504 |
| SRA |
SRX341646 |
