|
| Status |
Public on Mar 24, 2017 |
| Title |
hnRNP A1_iCLIP_zf_preZGA_rep2 |
| Sample type |
SRA |
| |
|
| Source name |
zebrafish embryo
|
| Organism |
Danio rerio |
| Characteristics |
developmental stage: preZGA (32 cell stage)
antibody: anti-hnRNP A1
barcode sequence: NNNTTGTNN
|
| Treatment protocol |
Zebrafish embryos at the desired developmental stages were irradiated with UV light (254 nm) to in vivo crosslink RNA-protein interactions.
|
| Growth protocol |
Zebrafish embryos were raised from WT AB strain and maintained using standard conditions.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Zebrafish embryos were lysed and lysates treated with RNaseI to obtain optimal length of RNA fragments for cDNA library preparation. hnRNP A1-RNA complexes were immunopurified using mouse anti-hnRNP A1 antibody and protein G-dynabeads (Invitrogen). Anti-IgG antibody (Sigma) was used for negative control immunopurifications.
After RNA dephosphorylation, 3' end linker ligation was performed. Protein-RNA complexes were resolved on SDS-PAGE and transferred to the nitrocellulose membrane. Region of the membrane above the predicted size of hnRNP A1 was cut and RNA extracted using proteise K treatment. Subsequently, RNA was reverse transcribed using RT primers harboring a barcode. cDNA was separated into three size selected fractions using 6% TBE-Urea gel (Life Technologies) and circularized by ssDNA circligase. Specific oligo was annealed for BamHI restriction site formation. cDNA was linearized by BamHI digestion. Final cDNA libraries were generated by PCR amplification.. For more details see: König et al. (2010): iCLIP reveals the function of hnRNP particles in splicing at individual nucleotide resolution. Nat Struct Mol Biol PMID:20601959
|
| |
|
| Library strategy |
OTHER |
| Library source |
transcriptomic |
| Library selection |
other |
| Instrument model |
Illumina HiSeq 2000 |
| |
|
| Description |
Each unique and multi mapping file contains significant CLIP tags (FDR<0.01) from the three summed replicates for their respective developental stages (preZGA and ZGA). Data provided for hnRNP A1 and IgG pull-downs.
peaks_id79493_rnd100_flank15_fdr0.01_group_3808_Replicates-together-hnRNPA1_sum_M_danRer7--ensembl72_from_3715-3716-3719_bedGraph-cDNA.bed.gz_lowFDR.bed
peaks_id79489_rnd100_flank15_fdr0.01_group_3808_Replicates-together-hnRNPA1_sum_G_danRer7--ensembl72_from_3715-3716-3719_bedGraph-cDNA-hits-in-genome_lowFDR.bed
|
| Data processing |
Basecalls performed using CASAVA version 1.4
For detail on data processing see: König et al. (2010): iCLIP reveals the function of hnRNP particles in splicing at individual nucleotide resolution. Nat Struct Mol Biol PMID:20601959
Genome_build: Zv9 (version 75)
Supplementary_files_format_and_content: .bed files describing significant crosslink sites derived from unique (contain G in the file name) and multi mapped (contain M in the file name) reads for combined replicates for each individual developmental stages and negative controls.
|
| |
|
| Submission date |
Dec 21, 2015 |
| Last update date |
May 15, 2019 |
| Contact name |
Karla M Neugebauer |
| E-mail(s) |
karla.neugebauer@yale.edu
|
| Organization name |
Yale University
|
| Department |
Molecular Biophysics and Biochemistry
|
| Street address |
333 Cedar St
|
| City |
New Haven |
| State/province |
CT |
| ZIP/Postal code |
06520 |
| Country |
USA |
| |
|
| Platform ID |
GPL14875 |
| Series (1) |
| GSE76212 |
Dynamic RNA-protein interactions underlie the zebrafish maternal-to-zygotic transition |
|
| Relations |
| BioSample |
SAMN04358610 |
| SRA |
SRX1494233 |
