Experiment ERX269875 Download

TitleMLE HITS-CLIP in Drosophila S2 cells
Design descriptionMLE HITS-CLIP in Drosophila S2 cells
Referencesno content
OrganismsDrosophila melanogaster
SubmitterEuropean Bioinformatics Institute (EBI)
InstrumentIllumina Genome Analyzer IIx
Library nameDNA
Library strategyOTHER
Library selectionother
Library layoutSINGLE
Library construction protocolS2 cells were grown in Schneiders Media (Invitrogen) supplemented with 10% of heat-inactivated bovine fetal serum to a density of 1 million cells per ml. HITS-CLIP was performed as described in (Chi et al., 2009) with the following modifications: Nuclear extracts prepared from Drosophila S2 cells were used for immunoprecipitations. After high-salt washes, the 3'-ends of bound RNAs were de-phosphorylated first with PNK in MES-PNK buffer (25mM MES at pH 6.0, 50mM NaCl, 10mM MgCl2, 0.1% Tween20, 5mM DTT, NO ATP, 1U/uL PNK [NEB]) by incubating the beads at 37 degrees C for 10 minutes and then with CIP (NEB) before ligation of the 3'-RNA tag. The libraries were prepared following Illumina's TruSeq ChIP sample prep kit (http://www.illumina.com/products/truseq_chip_sample_prep_kit.ilmn)
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Experimental Factor: immunoprecipitate anti-MLE antibody, long fragments