NextSeq 500 sequencing; ChIP-seq of neuroblastoma IMR-32, CLB-GA, NGP, IMR-5/75, GI-M-EN, CHP-212, and IMR-5 cell lines to identify TBX2, MYCN, H3K27ac, H3K4me1 and H3K4me3 binding sites
ChIP-seq of neuroblastoma IMR-32, CLB-GA, NGP, IMR-5/75, GI-M-EN, CHP-212, and IMR-5 cell lines to identify TBX2, MYCN, H3K27ac, H3K4me1 and H3K4me3 binding sites
A total of 3-5x10^7 cells were crosslinked in complete medium with 1% formaldehyde while shaking for 7 min at room temperature. Crosslinking was quenched with 125 nM glycine, cells were washed twice with PBS and stored at -80°C. Cells were lysed and sonicated with the M220 Covaris for 15 min to obtain 200-300 bp long fragments. Chromatin fragments were immunoprecipitated overnight using 1 ug antibody for every 10^7 cells. 20 ul Protein A UltraLink Resin beads was added per 10^7 cells. Beads were eluted by incubation for 22 min at 5°C while vortexing every 2 min, reverse crosslinking was done at 65°C for 15h. Antibodies used in this experiment are as follows: Goat Anti-TBX2 polyclonal antibody, Santa Cruz Biotechnology, Cat# sc-17880; RRID: AB_2200389;  Mouse Anti-N-MYC monoclonal antibody, Santa Cruz Biotechnology, Cat# sc-53993; RRID:AB_831602;  Rabbit Anti-H3K4me1 polyclonal antibody, Abcam Cat# ab8895, RRID:AB_306847; Rabbit Anti-H3K4me3 polyclonal antibody; Abcam; Cat# ab8580, RRID:AB_306649; Rabbit Anti-H3K27ac polyclonal antibody, Abcam Cat# ab4729,  RRID:AB_2118291  Cells were grown in RPMI1640 medium supplemented with 10% foetal bovine serum, 2mM L-Glutamine and 100 IU/ml penicillin/streptaviding at 37°C in a 5% CO2 humid atmosphere. The chromatin for DNA purposes was resuspended in TE-buffer to dilute SDS in the elution buffer, incubated for 2h at 37°C with 0.2 mg/ml RNase and followed by an incubation of 2h at 55°C with 0.2 mg/ml proteinase K. DNA was isolated using 400 ul phenol:chloroform:isoamylalcohol in phase lockd gel tubes. Upon centrifugation, the aqueous layer was transferred to a new tube with 200 mM NaCl, 30 ug glycogen and 800 ul 100% ethanol, and incubated for 30 min at -20°C. Upon centrifugation, the pellet was washed with 80% Ethanol and resuspended in RNase/DNase free water. Concentration was measured using the Qubit dsDNA HS Assay Kit. Library prep was done using the NEBNext Ultra DNA library Prep Kit from Illumina (E7370S) with 50-500 ng starting material and using 8-12 PCR cycles according to the manufacturer's instructions. Libraries were evaluated on the bio-analyzer (Agilent) using the Agilent high-sensitivity kit, followed by Pippin Prep with a 2% Dye Free Marker L Agarose Gel Cassette to remove large fragments or adapter dimers. Library concentrations were measured with the illumina Kapa Library quantification kit.
Experimental Factor: cell line
IMR-32
Experimental Factor: immunoprecipitate
anti-TBX2