title: MLE HITS-CLIP in Drosophila S2 cells
design: MLE HITS-CLIP in Drosophila S2 cells
library_construction_protocol: S2 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)
attributes.tag: Experimental Factor: immunoprecipitate
attributes.value: anti-MLE antibody, long fragments