organism: Human immunodeficiency virus
attributes.tag: Alias
attributes.value: E-MTAB-2700:iCLIP_GFP-A3G_HIV1-strainIIIB_packagedproteininvirions_Hs_NNNGGTCNN_20110811_LUe35_2
attributes.tag: Broker name
attributes.value: ArrayExpress
attributes.tag: Description
attributes.value: Protocols: CEM-SS T cell lines were grown in RPMI media supplemented with foetal bovine serum and Pen/Strept. HIV-1 viruses were produced from CEM-SS T cell lines by infecting cells with delta Vif IIIB strain HIV-1. HIV-1 viruses produced from CEM-SS T cell lines over expressing proteins of interest were concentrated and purified by ultracentrfugation through a sucrose gradient. Producer cells were washed throughly. Viruses or cells were then ressuspended in PBS and subjected to UV-C irradiation. Cells or viruses were ressuspended in 1mL of lysis buffer (50mM Tris-HCL, pH 7.4; 100mM NaCl; 1% NP-40; 0.1% SDS; 0.5% Sodium deoxycholate and protease inhibitor) and sonicated. 0.04ng of RNase A were added to the samples. RNAs cross-linked to proteins were extracted by immunoprecipitation of the proteins of interest iCLIP protocol was followed to isolate RNA bound to proteins of interest and amplify cDNA library. Proteins of interest were immunoprecipitated using anti-T7 or anti-GFP antibodies.. In order to differentiate between identical reads generated by PCR or distinct reads that were immunoprecipitated but that have the same sequence and length , we inserted 2 random nuclotides 5 prime of the barcode and 3 random nucleotides 3 prime of the barcode on each of the primers used to generate cDNA. Reads that have the same random nucleotides were judged to be artifacts from the PCR. This approach has been described in detail in Konig, et al, (2010): iCLIP reveals the function of hnRNP particles in splicing at individual nucleotide resolution, Nat Struct Mol Biol 17:909-915.. The final products were obtained by PCR with Illumina pair-end sequencing primers: 5-CAAGCAGAAGACGGCATACGAGATCGGTCTCGGCATTCCTGCTGAACCGCTCTTCCGATCT-3; 5-AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT-3
attributes.tag: ENA checklist
attributes.value: ERC000011
attributes.tag: INSDC center alias
attributes.value: EBI
attributes.tag: INSDC center name
attributes.value: European Bioinformatics Institute
attributes.tag: INSDC first public
attributes.value: 2014-12-17T17:02:21Z
attributes.tag: INSDC last update
attributes.value: 2014-07-16T16:22:51Z
attributes.tag: INSDC status
attributes.value: public
attributes.tag: SRA accession
attributes.value: ERS506239
attributes.tag: Sample Name
attributes.value: ERS506239
attributes.tag: Title
attributes.value: iCLIP_GFP-A3G_HIV1-strainIIIB_packagedproteininvirions_Hs_NNNGGTCNN_20110811_LUe35_2
attributes.tag: genotype
attributes.value: GFP-A3G packaging
attributes.tag: organism
attributes.value: Human immunodeficiency virus
attributes.tag: specimen with known storage state
attributes.value: fresh specimen
attributes.tag: strain
attributes.value: IIIB delta Vif