Illumina HiSeq 2000 sequencing; A deconvolution protocol for chip-seq reveals analogous enhancer structures on the mouse and human ribosomal RNA genes
A deconvolution protocol for chip-seq reveals analogous enhancer structures on the mouse and human ribosomal RNA genes
Cells were fixed in 1% formaldehyde for 8 min at room temperature. Nuclei were isolated using Lysis Buffer (10 mM Tris pH 7.5, 10 mM NaCl, 3 mM MgCl2, 0.5% NP-40), transferred to Sonication Buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 2 mM EGTA, 4 mM EDTA, 0.1% SDS, 1% Triton X-100, 1% NP-40) and sonicated (Bioruptor, Diagenode) for 30 cycles of 30 sec on / 30 sec off at high intensity. Each immunoprecipitation (IP) was carried out on the equivalent of 50 x 106 cells in IP Buffer (150 mM NaCl, 50 mM Tris-HCl pH 7.5, 5 mM EDTA, 0.5% NP-40, 1% Triton X-100) overnight at 4°C. The antibody slurry was prepared with 50 µl A-, 50 µl G-Dynabeads and 60 µg.ml-1 antibody per IP. See; Herdman et al. (2017). PLoS Genetics 13, e1006899. Rabbit polyclonal antibodies against mouse UBF, RPI large subunit (RPA194/Polr1A)were generated in the laboratory. Anti-RPA194 (#5) and anti-UBF (#8) antisera were raised against mouse RPA194 (aa 231–429) and mouse UBF (aa 2–404) ref: https://doi.org/10.1016/j.molcel.2010.03.015. K562 cells were grown to subconfluence in Iscove's Modified Dulbecco's Medium, 10% fetal bovine serum.  Immunoprecipitated chromatin was treated with RNaseA and the DNA isolated using 2% Na SDS and 2mg.ml-1 Proteinase-K. ChIP DNA samples were quality controlled by qPCR before being sent for library preparation and 50 base single-end sequencing on an Illumina HiSeq 2000 (McGill University and Genome Qubec Innovation Centre).
Experimental Factor: immunoprecipitate
anti mouse UBF (aa 2404)  replica 1