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Sample GSM4478387 Query DataSets for GSM4478387
Status Public on Aug 21, 2020
Title eclip_hela_ints11_abcam_IP_r2
Sample type SRA
 
Source name eclip_hela_ints11
Organism Homo sapiens
Characteristics cell line: HeLa
cell type: cervical adenocarcinoma
cell source: ATCC CCL-2, LOT# 63226283
chip antibody: INTS11 (Sigma prestige, HPA029025, LOT# A107128)
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.
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.
 
Library strategy RIP-Seq
Library source transcriptomic
Library selection other
Instrument model Illumina HiSeq 3000
 
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.
 
Submission date Apr 15, 2020
Last update date May 03, 2022
Contact name Felipe Beckedorff
E-mail(s) beckedorff@gmail.com
Organization name University of Miami
Street address 1501 NW 10th Avenue room 731.G , Miami, FL
City Miami
State/province FL
ZIP/Postal code 33136
Country USA
 
Platform ID GPL21290
Series (1)
GSE148755 Integrator restrains paraspeckles assembly by promoting isoform switching of the lncRNA NEAT1
Relations
BioSample SAMN14603682
SRA SRX8121018

Supplementary file Size Download File type/resource
GSM4478387_eclip_hela_ints11_abcam_IP_r2.neg.bw 7.4 Mb (ftp)(http) BW
GSM4478387_eclip_hela_ints11_abcam_IP_r2.peakClusters.normed.compressed.bed.gz 1.3 Mb (ftp)(http) BED
GSM4478387_eclip_hela_ints11_abcam_IP_r2.pos.bw 7.9 Mb (ftp)(http) BW
SRA Run SelectorHelp
Raw data are available in SRA
Processed data provided as supplementary file

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