title: GSM4478390: eclip_hela_igg_IP_r1; Homo sapiens; RIP-Seq
library_construction_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.
attributes.tag: GEO Accession
attributes.value: GSM4478390