 |
 |
GEO help: Mouse over screen elements for information. |
|
| Status |
Public on Feb 26, 2013 |
| Title |
LIN28A PAR-CLIP |
| Sample type |
SRA |
| |
|
| Source name |
HEK 293
|
| Organism |
Homo sapiens |
| Characteristics |
crosslinker and crosslinking wavelength: 4-SU / 365 nm
antibody: Flag M2
immunoprecipitated protein: LIN28A
|
| Treatment protocol |
For UV crosslinking, cells were washed once with ice-cold PBS while still attached to the plates. PBS was removed completely and cells were irradiated on ice with 365 nm UV light in a Stratalinker 2400 (Stratagene). Cells were scraped off with a rubber policeman in 1 ml PBS per plate and collected by centrifugation at 500×g for 5 min.
|
| Growth protocol |
HEK293 T-REx Flp-In cells (Invitrogen) were grown in D-MEM high glucose with 10% (v/v) fetal bovine serum, 1% (v/v) 2 mM L-glutamine, 1% (v/v) 10,000 U/ml penicillin/10,000 µg/ml streptomycin, 100 µg/ml zeocin and 15 µg/ml blasticidin. Cell lines stably expressing FLAG/HA-tagged LIN28A or B were generated by co-transfection of pFRT/TO/FLAG/HA/LIN28A or pFRT/TO/FLAG/HA/LIN28B construct with pOG44 (Invitrogen). Cells were selected by exchanging zeocin with 100 µg/ml hygromycin. Expression of FLAG/HA-LIN28A or FLAG/HA-LIN28B was induced by addition of 250 ng/ml doxycycline 15 to 20 h before crosslinking.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
The pellets of cells crosslinked with UV 365 nm were resuspended in NP40 lysis buffer and incubated on ice for 10 min. The cell lysate was cleared by centrifugation at 13,000g. RNase T1 (Fermentas) was added to the cleared cell lysates and the reaction mixture was incubated at 22ºC for 15 min and subsequently cooled for 5 min on ice before addition of antibody-conjugated magnetic beads. The recovered RNA was carried through a cDNA library preparation protocol originally described for cloning of small regulatory RNAs (Hafner et al., 2008). The first step, 3' adapter ligation, was carried out as described on a 20 ul scale using 10.5 ul of the recovered RNA. UV 365 nm crosslinked sample RNAs were processed using Solexa sequencing adapter sets. Depending on the amount of RNA recovered, 5'-adapter-3'-adapter products without inserts may be detected after amplification of the cDNA as additional PCR bands. In such case, the longer PCR product of expected size was excised from a 3% NuSieve low-melting point agarose gel, eluted from the gel pieces with the Illustra GFX-PCR purification kit (GE Healthcare) and Solexa sequenced.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2000 |
| |
|
| Description |
LIN28A ribonucleoprotein complexes
LIN28A.xlsx
|
| Data processing |
Mapping Reads: The reads from the LIN28A and B PAR-CLIP deep sequencing library were stripped of the 3’ adapter sequence using the FASTX toolkit (http://hannonlab.cshl.edu/fastx_toolkit/). Reads that were less than 20 nt in length or contained an ambiguous nucleotide were discarded. The remaining reads were aligned to the human genome (hg19), with up to 1 mismatch allowed, by the Bowtie algorithm (Langmead et al., 2009). Mapped locations were only reported for those with the minimum number of observed mismatches for each read. All T to C mismatches between the RNA fragment and the genome were subtracted from the mismatch count for each mapped location. After subtraction only reads that mapped to a single genomic location at the minimum number of mismatches were used for further analysis. The location that a read mapped, relative to a known transcript, was determined based on the ENSEMBL v57 database 1(Hubbard et al., 2007). If a read mapped to a location representing multiple categories, it was reported to belong to the category based on the following order of preference: 3’UTR, coding sequence, 5’UTR, intron, non-coding RNA, intergenic. Defining HuR binding sites by PARalyzer: Overlapping reads were grouped together for further analysis. A group must have contained ≥5 reads with T-to-C conversions at two or more locations and at least 25% of mapped sequence reads. PARalyzer is available at http://www.genome.duke.edu/labs/ohler/research/PARalyzer
Genome_build: hg19
|
| |
|
| Submission date |
Feb 25, 2013 |
| Last update date |
May 15, 2019 |
| Contact name |
Miguel Brown |
| E-mail(s) |
miguel.a.brown@gmail.com
|
| Organization name |
The Rockefeller University
|
| Street address |
1230 York Ave Box 186
|
| City |
New York |
| State/province |
New York |
| ZIP/Postal code |
10065 |
| Country |
USA |
| |
|
| Platform ID |
GPL11154 |
| Series (2) |
| GSE44615 |
Identification of mRNAs bound and regulated by human LIN28 proteins and molecular requirements for RNA recognition [PAR-CLIP] |
| GSE44616 |
Identification of mRNAs bound and regulated by human LIN28 proteins and molecular requirements for RNA recognition |
|
| Relations |
| SRA |
SRX245353 |
| BioSample |
SAMN01924688 |
| Supplementary data files not provided |
SRA Run Selector |
| Processed data are available on Series record |
| Raw data are available in SRA |
|
|
|
|
 |
