iCLIP_hnRNPH_RON_G305A
Homo sapiens
Protocols: Media from cells growing in a 6-well tissue culture plate was replaced with 1 ml cold PBS and the plate was put on ice. Next, the cells were irradiated once with 150 mJ/cm2 in a Stratalinker 2400 at 254 nm. Then the cells were harvested by scraping and collected by centrifugation at 400xg for 1 min at 4 °C. The supernatant was subsequently removed and the cell pellets were either snap-frozen and stored or immediately subjected to immunoprecipitation. One well of a 6-well tissue culture with 90% confluent cells were each transfected with 2 µg of a a G305A Mutant RON minigene one day prior to harvesting using 20 µg polyethylenimine MW ~ 2500 transfection reagent (Polysciences, Inc.). The RON minigene was cloned into the pcDNA 3.1 (+) vector (Invitrogen). The minigene construct contains a sequence of the MST1R gene including exon 11 together with the complete flanking introns and the constitutive exons 10 and 12 (chr3: 49,933,098 - 49,933,837, GRCh37/hg19). The RON minigene library comprises of approximately 6,000 minigenes with three to four mutations on average per minigene. For the iCLIP experiments, however, we transfected individual minigene variants (a wild type minigene and a point mutation plasmid G305A). Protein-RNA complexes were immunoprecipitated by using 7.5 µg of a polyclonal rabbit anti-HNRNPH antibody from Abcam (AB10374). Then, RNase digestion was performed by adding 10 µl of 1/100 diluted RNase I (Ambion) to each sample. Further processing of the sample including RNA extraction was performed according to a previously published protocol (Sutandy F.R., Hildebrandt A., König J., 2016). cDNA synthesis was carried out using the Superscript III (Life Technologies) reverse transcriptase according to the manufacturer's protocol. Subsequently, the cDNA was gel-purified and circularized according to a previously published protocol (Sutandy F.R., Hildebrandt A., König J., 2016). Next, the cDNA was PCR-amplified and the products were purified using the MinElute PCR Purification Kit from Qiagen. Finally, DNA fragments of length 150 - 225 bp were isolated using the DNA 500 LabChip Kit from Perkin Elmer according to the manufacturer's instructions.
ENA first public
2018-06-27
ENA last update
2017-11-22
External Id
SAMEA104396540
INSDC center alias
Institute of Molecular Biology (IMB)
INSDC center name
Institute of Molecular Biology (IMB)
INSDC first public
2018-06-27T17:02:54Z
INSDC last update
2017-11-22T11:06:12Z
INSDC status
public
Submitter Id
E-MTAB-6221:iCLIP_hnRNPH_RON_G305A
broker name
ArrayExpress
cell line
HEK293T
cell type
epithelial cell
common name
human
developmental stage
late embryo
disease
normal
genotype
MST1R G305A point mutant minigene construct
organism part
kidney
sample name
E-MTAB-6221:iCLIP_hnRNPH_RON_G305A