LINT - a novel dL(3)mbt containing complex represses malignant brain tumour signature genes

ERX032305 E-MTAB-855:SEQ CHIP_S2_dLint1 LINT - a novel dL(3)mbt containing complex represses malignant brain tumour signature genes - sequencing data ERP001006 E-MTAB-855 LINT - a novel dL(3)mbt containing complex represses malignant brain tumour signature genes - sequencing data ERS071646 E-MTAB-855:S2_dLint1 CHIP_S2_dLint1 ChIP-Seq GENOMIC ChIP D. melanogaster cell lines were maintained under standard conditions. ChIPs were performed as described in the Upstate Biotechnology ChIP assay protocol. 100*10^6 Kc cells were fixed in 1% formaldehyde for 10 min at RT. Fixation was stopped by the addition of 240 mM glycine. Cells were harvested, washed in ice cold PBS, resuspended in 1 ml SDS-Lysis buffer (50 mM Tris-HCl, pH 8.0, 10 mM EDTA, 1% SDS, protease inhibitors) and incubated for 10 min on ice. Lysates were sonicated using a Bioruptor (Diagenode) to obtain an average fragment length of 0.5 kb and centrifuged (at 4 degrees C, 15 min, 13000 rpm). Shearing of the DNA was analyzed by agarose gel electrophoresis following reversal of crosslinks. The supernatant (chromatin) was subjected to ChIP analysis. 8 microliter of anti-dLint-1 #1 were used per ChIP. 140 microliter of chromatin were used per ChIP, diluted 10x with IP-buffer (16.7 mM Tris-HCl, pH 8.0, 16.7 mM NaCl, 1.2 mM EDTA, 0.01% SDS, 1.1% Triton X-100, protease inhibitors) and pre-cleared with 40 microliter of pre-blocked ProtG beads (1mg/ml BSA, 4 h) (GE Healthcare) for 30 min with rotation. Incubation with antibodies was performed overnight, prior to incubation for 2 h with 35 microliter of 1:1 ProtG slurry at 4 degrees C. Precipitates were serially washed for 10 min, three times with low salt wash buffer (20 mM Tris-HCl pH 8.0, 150 mM NaCl, 2 mM EDTA, 0.1% SDS, 1% Triton-X-100), three times with high salt wash buffer (20 mM Tris-HCl pH 8.0, 500 mM NaCl, 2 mM EDTA, 0.1% SDS, 1% Triton-X-100), once with LiCl wash buffer (10 mM Tris pH 8.0, 250 mM LiCl, 1 mM EDTA, 1% NP-40, 1% sodium deoxycholate), once with TE buffer. Immunoprecipitates were eluted twice with 250 microliter elution buffer (1% SDS, 0.1 M NaHCO3) for each 15 min at RT and crosslinks were reversed by addition of 20 microliter 5 M NaCl and heating at 65 degrees C overnight. Following addition of 10 microliter of 0.5 M EDTA, 20 microliter 1 M Tris-HCl, pH 6.5 and 2 microliter of 10 mg/ml Proteinase K samples were incubated for 1 h at 45 degrees C. DNA was purified with peqGOLD Cycle-Pure Kit (Peqlab) Generation of Lint-1 specific antibodies The C-terminus (aa 302-602) of dLint-1 was fused to GST by cloning into pGex4T1 vector. The recombinant protein was expressed in E. coli BL21 according to standard procedures. The GST-Lint-1-C-term fusion protein was purified via affinity chromatography using a GSTrap FF column (GE Healthcare) and subsequent ion exchange chromatography using a HiTrap SP HP column (GE Healthcare) on an Akta purifier system (GE Healthcare) according to the manufacturer's instructions. For immunization two rabbits (serum #1 and #2) were injected with 0.5 mg of purified GST-Lint-1-C-term fusion protein each (Peptide Speciality Laboratories, Heidelberg, Germany). The specificity of antibodies was verified by RNA interference in Kc cells and subsequent Western blot analysis of nuclear extracts. Illumina Genome Analyzer IIx Experimental Factor: CELL_LINE S2 ERX032306 E-MTAB-855:SEQ CHIP_KC_dLint1 LINT - a novel dL(3)mbt containing complex represses malignant brain tumour signature genes - sequencing data ERP001006 E-MTAB-855 LINT - a novel dL(3)mbt containing complex represses malignant brain tumour signature genes - sequencing data ERS071647 E-MTAB-855:KC_dLint1 CHIP_KC_dLint1 ChIP-Seq GENOMIC ChIP D. melanogaster cell lines were maintained under standard conditions. ChIPs were performed as described in the Upstate Biotechnology ChIP assay protocol. 100*10^6 Kc cells were fixed in 1% formaldehyde for 10 min at RT. Fixation was stopped by the addition of 240 mM glycine. Cells were harvested, washed in ice cold PBS, resuspended in 1 ml SDS-Lysis buffer (50 mM Tris-HCl, pH 8.0, 10 mM EDTA, 1% SDS, protease inhibitors) and incubated for 10 min on ice. Lysates were sonicated using a Bioruptor (Diagenode) to obtain an average fragment length of 0.5 kb and centrifuged (at 4 degrees C, 15 min, 13000 rpm). Shearing of the DNA was analyzed by agarose gel electrophoresis following reversal of crosslinks. The supernatant (chromatin) was subjected to ChIP analysis. 8 microliter of anti-dLint-1 #1 were used per ChIP. 140 microliter of chromatin were used per ChIP, diluted 10x with IP-buffer (16.7 mM Tris-HCl, pH 8.0, 16.7 mM NaCl, 1.2 mM EDTA, 0.01% SDS, 1.1% Triton X-100, protease inhibitors) and pre-cleared with 40 microliter of pre-blocked ProtG beads (1mg/ml BSA, 4 h) (GE Healthcare) for 30 min with rotation. Incubation with antibodies was performed overnight, prior to incubation for 2 h with 35 microliter of 1:1 ProtG slurry at 4 degrees C. Precipitates were serially washed for 10 min, three times with low salt wash buffer (20 mM Tris-HCl pH 8.0, 150 mM NaCl, 2 mM EDTA, 0.1% SDS, 1% Triton-X-100), three times with high salt wash buffer (20 mM Tris-HCl pH 8.0, 500 mM NaCl, 2 mM EDTA, 0.1% SDS, 1% Triton-X-100), once with LiCl wash buffer (10 mM Tris pH 8.0, 250 mM LiCl, 1 mM EDTA, 1% NP-40, 1% sodium deoxycholate), once with TE buffer. Immunoprecipitates were eluted twice with 250 microliter elution buffer (1% SDS, 0.1 M NaHCO3) for each 15 min at RT and crosslinks were reversed by addition of 20 microliter 5 M NaCl and heating at 65 degrees C overnight. Following addition of 10 microliter of 0.5 M EDTA, 20 microliter 1 M Tris-HCl, pH 6.5 and 2 microliter of 10 mg/ml Proteinase K samples were incubated for 1 h at 45 degrees C. DNA was purified with peqGOLD Cycle-Pure Kit (Peqlab) Generation of Lint-1 specific antibodies The C-terminus (aa 302-602) of dLint-1 was fused to GST by cloning into pGex4T1 vector. The recombinant protein was expressed in E. coli BL21 according to standard procedures. The GST-Lint-1-C-term fusion protein was purified via affinity chromatography using a GSTrap FF column (GE Healthcare) and subsequent ion exchange chromatography using a HiTrap SP HP column (GE Healthcare) on an Akta purifier system (GE Healthcare) according to the manufacturer's instructions. For immunization two rabbits (serum #1 and #2) were injected with 0.5 mg of purified GST-Lint-1-C-term fusion protein each (Peptide Speciality Laboratories, Heidelberg, Germany). The specificity of antibodies was verified by RNA interference in Kc cells and subsequent Western blot analysis of nuclear extracts. Illumina Genome Analyzer IIx Experimental Factor: CELL_LINE KC