|
| Status |
Public on Oct 20, 2016 |
| Title |
hnRNPA2/B1_eCLIP_004 |
| Sample type |
SRA |
| |
|
| Source name |
iPSC derived motor neurons
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: CTRL.JCV
age: 28 days
tissue type: iPSC-MNs
antibody: null
vendor: null
catalog number: null
lot number: null
|
| Treatment protocol |
none
|
| Growth protocol |
CTRL.JCV iPSCs were differentiated into motor neurons as described. Samples were harvested 28 days post neural induction.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
10cm dishes of motor neuros were rinsed in PBS and UV crosslinked. Crosslinked cells were peletted and snap frozen in liquid nitrogen
eCLIP libraries were prepared as previously described (Van Nostrand et al. 2016) using rabbit polyclonal antibody GTX127928 (Genetex) (10 mg per sample). Human iPSC-MNs were differentiated in culture for 28 days. Approximately 20 million cells were UV crosslinked at λ=254nm with 4000 mJ/cm2 of energy. Crosslinked cell pellets were lysed and treated with RNAse I (Ambion). Lysate was immunoprecipitated using rabbit polyclonal antibody GTX127928 (Genetex) (10 g per sample) and Dynabeads Protein G (Thermo Fisher Scientific). Immunoprecipitated samples were washed and dephosphorylated with Fast AP (Thermo Fisher Scientific). 3’ phosphates were applied using T4 PNK (NEB) and a 3’ RNA adapter was ligated using T4 RNA Ligase (NEB). Samples were run on PAGE gels and transferred to nitrocellulose membranes. A region of membrane spanning 75kDa above the molecular weight of hnRNP A2/B1 was cut and RNA was recovered using Proteinase K and phenol-chloroform. RNA was reverse transcribed using Affinityscript (Agilent Technologies) and treated with ExoSAP-IT (Affymetrix). A DNA adapter containing a 5nt randomer was then ligated to the 3’ end (T4 RNA Ligase, NEB). Samples were purified using Dynabeads MyOne Silane (Thermo Fisher Scientfic), quantified by qPCR, and then PCR amplified to the appropriate number of cycles. Amplified libraries were size selected with an agarose gel, quantified using the Agilent Tapestation, and sequenced on the Illumina HiSeq 2500 platform using paired end 50bp reads.
|
| |
|
| Library strategy |
RIP-Seq |
| Library source |
transcriptomic |
| Library selection |
other |
| Instrument model |
Illumina HiSeq 2500 |
| |
|
| Description |
eCLIP RNA
|
| Data processing |
Reads were demultiplexed using custom scripts and the randomer was appended to the read name. Reads were trimmed, filtered for repetitive elements, and mapped to human genome assembly hg19 as described in iCLIP computational analysis. PCR duplicated reads were removed based on the start positions of read1, read2, and the sequence of the randomer. eCLIP peaks were identified using CLIPPER with parameters –s hg19 –o –bonferroni –superlocal --threshold-method binomial --save-pickle (Lovci et al. NSMB, 2013). Peak strength was then normalized against a size matched input by calculating fold enrichment of number of reads in IP versus number of reads in size matched input. Peaks were called significant if the number of reads in IP was greater than the number of reads in input and the the peaks a Bonferroni corrected fisher exact p-value of less than .05.
Genome_build: hg19
Supplementary_files_format_and_content: peaks.bed and bigwig
|
| |
|
| Submission date |
Aug 25, 2016 |
| Last update date |
May 15, 2019 |
| Contact name |
Gene Yeo |
| E-mail(s) |
geneyeo@ucsd.edu
|
| Organization name |
UCSD
|
| Street address |
2880 Torrey Pines Scenic Dr. Room 3805/Yeo Lab
|
| City |
La Jolla |
| State/province |
CA |
| ZIP/Postal code |
92037 |
| Country |
USA |
| |
|
| Platform ID |
GPL16791 |
| Series (2) |
| GSE86040 |
HNRNPA2B1 regulates alternative RNA processing in the nervous system and accumulates in granules in ALS IPSC-derived motor neurons [hnRNPA2B1_eCLIP_human_iPSC_MN] |
| GSE86464 |
HNRNPA2B1 regulates alternative RNA processing in the nervous system and accumulates in granules in ALS IPSC-derived motor neurons |
|
| Relations |
| BioSample |
SAMN05730920 |
| SRA |
SRX2140555 |
