Name: CHIP_KC_dLint1
Instrument: Illumina Genome Analyzer IIx
Strategy: ChIP-Seq
Source: GENOMIC
Selection: ChIP
Layout: SINGLE
Construction protocol: D. melanogaster cell lines were maintained under standard conditions. ChIPs were performed as described in the Upstate Biotechnology ChIP assay<br>protocol. 100*10^6 Kc cells were fixed in 1% formaldehyde for 10 min at RT.<br>Fixation was stopped by the addition of 240 mM glycine. Cells were harvested,<br>washed in ice cold PBS, resuspended in 1 ml SDS-Lysis buffer (50 mM Tris-HCl,<br>pH 8.0, 10 mM EDTA, 1% SDS, protease inhibitors) and incubated for 10 min on<br>ice. Lysates were sonicated using a Bioruptor (Diagenode) to obtain an average<br>fragment length of 0.5 kb and centrifuged (at 4 degrees C, 15 min, 13000 rpm).<br>Shearing of the DNA was analyzed by agarose gel electrophoresis following<br>reversal of crosslinks. The supernatant (chromatin) was subjected to ChIP<br>analysis. 8 microliter of anti-dLint-1 #1 were used per ChIP. 140 microliter of<br>chromatin were used per ChIP, diluted 10x with IP-buffer (16.7 mM Tris-HCl, pH<br>8.0, 16.7 mM NaCl, 1.2 mM EDTA, 0.01% SDS, 1.1% Triton X-100, protease<br>inhibitors) and pre-cleared with 40 microliter of pre-blocked ProtG beads<br>(1mg/ml BSA, 4 h) (GE Healthcare) for 30 min with rotation. Incubation with<br>antibodies was performed overnight, prior to incubation for 2 h with 35<br>microliter of 1:1 ProtG slurry at 4 degrees C. Precipitates were serially<br>washed for 10 min, three times with low salt wash buffer (20 mM Tris-HCl pH<br>8.0, 150 mM NaCl, 2 mM EDTA, 0.1% SDS, 1% Triton-X-100), three times with high<br>salt wash buffer (20 mM Tris-HCl pH 8.0, 500 mM NaCl, 2 mM EDTA, 0.1% SDS, 1%<br>Triton-X-100), once with LiCl wash buffer (10 mM Tris pH 8.0, 250 mM LiCl, 1 mM<br>EDTA, 1% NP-40, 1% sodium deoxycholate), once with TE buffer.<br>Immunoprecipitates were eluted twice with 250 microliter elution buffer (1%<br>SDS, 0.1 M NaHCO3) for each 15 min at RT and crosslinks were reversed by<br>addition of 20 microliter 5 M NaCl and heating at 65 degrees C overnight.<br>Following addition of 10 microliter of 0.5 M EDTA, 20 microliter 1 M Tris-HCl,<br>pH 6.5 and 2 microliter of 10 mg/ml Proteinase K samples were incubated for 1 h<br>at 45 degrees C. DNA was purified with peqGOLD Cycle-Pure Kit (Peqlab)<br><br>Generation of Lint-1 specific antibodies<br>The C-terminus (aa 302-602) of dLint-1<br>was fused to GST by cloning into pGex4T1 vector. The recombinant protein was<br>expressed in E. coli BL21 according to standard procedures. The<br>GST-Lint-1-C-term fusion protein was purified via affinity chromatography using<br>a GSTrap FF column (GE Healthcare) and subsequent ion exchange chromatography<br>using a HiTrap SP HP column (GE Healthcare) on an Akta purifier system (GE<br>Healthcare) according to the manufacturer's instructions. For immunization two<br>rabbits (serum #1 and #2) were injected with 0.5 mg of purified<br>GST-Lint-1-C-term fusion protein each (Peptide Speciality Laboratories,<br>Heidelberg, Germany). The specificity of antibodies was verified by RNA<br>interference in Kc cells and subsequent Western blot analysis of nuclear<br>extracts.