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Status |
Public on Aug 21, 2020 |
Title |
eclip_hela_igg_IP_r1 |
Sample type |
SRA |
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Source name |
eclip_hela_igg_IP_r1
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Organism |
Homo sapiens |
Characteristics |
cell line: HeLa cell type: cervical adenocarcinoma cell source: ATCC CCL-2, LOT# 63226283 chip antibody: rabbit IgG (Invitrogen, 02-6102, LOT# R1238244)
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Growth protocol |
HeLa cells were maintained at 37°C with 5% CO2 and passaged every 2-3 days at a 1:3 split ratio, according to ATCC recommendations. DMEM medium was supplemented with 10% FBS.
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Extracted molecule |
total RNA |
Extraction protocol |
eCLIP was performed according to Van Nostrand, E., Pratt, G., Shishkin, A. et al. Robust transcriptome-wide discovery of RNA-binding protein binding sites with enhanced CLIP (eCLIP). Nat Methods 13, 508–514 (2016). https://doi.org/10.1038/nmeth.3810 libraries were prepared as described in Van Nostrand, E., Pratt, G., Shishkin, A. et al. Robust transcriptome-wide discovery of RNA-binding protein binding sites with enhanced CLIP (eCLIP). Nat Methods 13, 508–514 (2016). https://doi.org/10.1038/nmeth.3810 In brief, 2 x 107 cells were crosslinked by UV-C irradiation (254 nm, 400 mJ/cm2) and lysed on ice followed by sonication. Antibodies were incubated with Dynabeads™ M-280 Sheep Anti-Rabbit IgG (Invitrogen, 11204D) for 1 hour. After limiting RNase I (Ambion) digest in presence of DNase, the lysate was subjected to immunoprecipitation at 4˚C for 16 hours. In the following, 2% of the lysate was removed for size-matched input control. IP efficiency and specificity were verified by immunoblot using 20% of the IP material. Co-immunoprecipitated RNA was dephosphorylated, followed by 3’RNA adapter ligation using T4 RNA Ligase (NEB). Input and IgG controls and INTS11-RNA complexes were run on a NuPAGETM 4-12% Bis-Tris Gel, transferred to nitrocellulose and cut from the membrane between 65-145kDa. Protein-bound RNA was released from the membrane by Urea/ Proteinase K digest, followed by acid Phenol/ Chloroform/ Isoamyl alcohol RNA extraction and purification using RNA Clean & Concentrator (Zymo Research). After reverse transcription (AffinityScript reverse transcriptase, Agilent), RNA was treated with exonuclease (ExoSAP-IT, Affymetrix) and removed by combined NaOH/ HCl treatment. A 3’Linker was ligated to the cDNA, and the resulting library was PCR amplified using Q5 Polymerase (NEB), purified and size selected for sequencing. Single-end (SE100) sequencing was performed to an average of 40 million reads per sample using Illumina HiSeq 3000 sequencer.
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Library strategy |
RIP-Seq |
Library source |
transcriptomic |
Library selection |
other |
Instrument model |
Illumina HiSeq 3000 |
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Data processing |
Data was processed according to Van Nostrand, E., Pratt, G., Shishkin, A. et al. Robust transcriptome-wide discovery of RNA-binding protein binding sites with enhanced CLIP (eCLIP). Nat Methods 13, 508–514 (2016). https://doi.org/10.1038/nmeth.3810 https://github.com/YeoLab/eclip Genome_build: hg19 Supplementary_files_format_and_content: bigwig files, strand specific bed files: clipper v1.1 output (col1: chrom, col2: chromStart, col3: chromEnd, col4: -log10 pvalue, col5: log2 fold enrichment above input, col6: strand)) contains clusters of Ints11 binding.
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Submission date |
Apr 15, 2020 |
Last update date |
May 03, 2022 |
Contact name |
Felipe Beckedorff |
E-mail(s) |
beckedorff@gmail.com
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Organization name |
University of Miami
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Street address |
1501 NW 10th Avenue room 731.G , Miami, FL
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City |
Miami |
State/province |
FL |
ZIP/Postal code |
33136 |
Country |
USA |
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Platform ID |
GPL21290 |
Series (1) |
GSE148755 |
Integrator restrains paraspeckles assembly by promoting isoform switching of the lncRNA NEAT1 |
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Relations |
BioSample |
SAMN14603679 |
SRA |
SRX8121021 |
Supplementary file |
Size |
Download |
File type/resource |
GSM4478390_eclip_hela_igg_IP_r1.neg.bw |
274.4 Kb |
(ftp)(http) |
BW |
GSM4478390_eclip_hela_igg_IP_r1.pos.bw |
290.4 Kb |
(ftp)(http) |
BW |
SRA Run Selector |
Raw data are available in SRA |
Processed data provided as supplementary file |
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