Illumina Genome Analyzer IIx sequencing; ChIP-seq analysis of human neuroblastoma cell lines
ChIP-seq analysis of human neuroblastoma cell lines
Cell line   SH-SY5Y/6TR(EU)/pTrex-Dest-30/MYCN (SY5Y-MYCN) was maintained in RPMI 1640 media (Gibco) supplemented with 10% fetal bovine serum (FBS) (Gibco) and 1% Penicillin-Streptomycin solution (Gibco). Transgenic cell line SY5Y-MYCN was maintained in the same media as for other NB cells and supplemented with G418 (0.2 mg/ml) (Sigma-Aldrich) and Blasticitidin (7.5 g/ml) (Invitrogen). Induction of SY5Y-MYCN cells (24h and 48h samples) was performed by adding Doxycycline hyclate (Sigma-Aldrich) at the final concentration of 1 g/ml. Cells were cultivated in cell culture incubator (Thermo Scientific) at 37oC and 5% CO2. Cells were passaged before reaching confluence of not more than 90%, every 3-5 days depending on the cell type and seeding density. Sub-cultivation of cells was performed by aspiration of the media and adding Versene (1x PBS supplemented with 0.5 mM EDTA). ChIP sample preparation was performed using SimpleChIP Enzymatic Chromatin IP Kit (Agarose Beads) by Cell Signalling. Brief description of the protocol is as follows. To crosslink proteins to DNA, 540 l of 37% formaldehyde (Sigma-Aldrich) was added to each 15 cm culture dish with NB cells (90% confluency; 10 million cells per dish approximately) containing 20 ml medium. Upon gentle swirling cells are incubated for 10 minutes at room temperature. Then 2 ml of 10X glycine was added to each 15 cm dish, briefly mixed and incubated for 5 minutes at room temperature. Media was removed and cells were washed two times with 20 ml ice-cold 1x PBS, with completely removing wash from culture dish each time. Then 2 ml of ice-cold 1X PBS + PMSF was added to the 15 cm dish. Cells were scraped and resuspended into cold buffer. Combined plates (2 per sample) were placed in15 ml conical tube and centrifuged at 1,500 rpm in a bench top centrifuge for 5 min at 4C.Upon removing supernatant, resuspended cells (10 ml ice-cold Buffer A + DTT + Protease Inhibitor Cocktail + PMSF) were incubated on ice for 10 min and mixed by inverting every 3 min. Nuclei were pelleted by centrifugation at 3000 rpm in a bench top centrifuge for 5 min at 4C. Supernatant was removed and pellet was resuspended in 10 ml ice-cold Buffer B + DTT. This step was followed by repeated centrifugation, removing supernatant, and resuspension of pellet in 1.0 ml Buffer B + DTT. Sample was transferred to a 1.5 ml microcentrifuge tube and then incubated with 2.5 l of Micrococcal Nuclease for 20 min at 37C with frequent mixing to digest DNA to length of approximately 150-900 bp. Digestion was stopped by adding 100 l of 0.5 M EDTA and placing tube on ice. Nuclei were pelleted by centrifugation at 13,000 rpm in a microcentrifuge for 1 min at 4C which was followed by removing of supernatant. Nuclear pellet was resuspended in 1 ml of 1X ChIP buffer + PIC + PMSF, split into two tubes of 500 l and incubated on ice for 10 min. Each tube was sonicated with 3 pulses of 10 seconds with 30 sec breaks using Bransonic Sonicator. Lysates were clarified by centrifugation at 10,000 rpm in a microcentrifuge for 10 min at 4C and supernatant was transferred to a new tube. This is the cross-linked chromatin preparation, which was stored at -80C until further use. 10 g of the crosslinked chromatin was added to 400 l of the ChIP buffer (Cell Signalling) for each immunoprecipitation. At this stage, a 10 l sample was removed for the 2% Input Sample. For each immunoprecipitation, 500 l of the diluted chromatin was transferred to a microcentrifuge tube and immunoprecipitating antibody was added. For the positive control, 10 l of Histone H3 (D2B12) XP Rabbit mAb (ChIP Formulated) #4620 was added to the IP sample. 20 l of the MYCN antibody or 2 l of RNA PolII antibodies were added. For the negative control 1 l of normal rabbit or mouse IgG were added to the IP sample. IP samples were incubated overnight at 4C with rotation. Next day 30 l of ChIP-Grade Protein G Agarose Beads were added and incubated for 2 h at 4C with rotation. Protein G Agarose Beads were pelleted in each immunoprecipitation by brief 1 min centrifugation at 6000 rpm, followed by the removal of the supernatant. 1 ml of Low Salt Wash buffer (Cell Signalling) was added to the beads and incubated at 4C for 5 min with rotation. This step was repeated two more times. 1 ml of High Salt Wash buffer (Cell Signalling) was added to the beads and incubated at 4C for 5 min with rotation. Protein G Agarose Beads were pelleted by brief 1 min centrifugation at 6000 rpm and supernatant was removed. 150 l of 1X ChIP Elution Buffer was added to each IP sample. Elution of chromatin from the antibody/Protein G beads was performed for 30 minutes at 65C with gentle vortexing (1,200 rpm) in the thermomixer (Eppendorf). Then, Protein G Agarose Beads were pelleted by brief 1 min centrifugation at 6,000 rpm and eluted chromatin in the supernatant was placed to a new tube. Reverse cross-linking was achieved by adding 6 l 5M NaCl and 2 l Proteinase K, and incubating samples for 2 h at 65C. DNA purification was performed using QiaQuick PCR Purification (Qiagen) columns according to the manufacturers instructions.  Library preparation for ChIP-seq samples was performed using the NEXTflexTM ChIP-Seq Kit ( Bioo Scientific). We have utilised the protocol for the single end multiplexed genomic DNA sequencing compatible with using Illumina GAIIX and HiSeq platforms. The Library Prep kit is based on a series of proprietary Master Mix reagents and magnetic bead based clean-up for the appropriate size exclusion, adapter ligation and amplification. All libraries were prepared using 10 ng of quality assessed ChIP DNA. Detailed protocol for the Library preparation was strictly followed. Full details of the protocol are available at: http://www.biooscientific.com/Portals/0/Manuals/NGS/5143-01-NEXTflex-ChIP-Seq-Kit.pdf.
Experimental Factor: MYCN
immunoprecipitate
Experimental Factor: MYCN-tet-off
genotype