TTF1 control of LncRNA synthesis and cell growth delineates a tumour suppressor pathway acting directly on the  ribosomal RNA Genes
TTF1 control of LncRNA synthesis and cell growth delineates a tumour suppressor pathway acting directly on the  ribosomal RNA Genes
Cells were fixed with 1% formaldehyde for 8 min at room temperature. Formaldehyde was quenched by addition of 125 mM Glycine and cells harvested and washed in PBS. Nuclei were isolated using an ultrasound-based nuclei extraction method (NEXSON: Nuclei Extraction by SONication) (ref: doi: 10.1093/nar/gkv1495. PubMed PMID: 26704968) with some modifications. Briefly, for all cell types, 33 million cells were resuspended in 1.5 ml of Farnham lab buffer (5 mM PIPES pH 8.0, 85 mM KCl, 0.5% IGEPAL, protease inhibitors). Cell suspensions were sonicated in 15 ml polystyrene tubes (BD #352095) using 3 to 4 cycles of 15 sec on : 30 sec off at low intensity in a Bioruptor (Diagenode). After recovery of the NEXSON-isolated nuclei by centrifugation (1000g, 5 min), nuclei were resuspended in 1.5 ml of shearing buffer (10 mM Tris-HCl pH 8.0, 1 mM NaEDTA, 0.1% SDS, protease inhibitors) and sonicated for 25 min, 30 sec on : 30 sec off , at high intensity. Each immunoprecipitation (IP) was carried out using the equivalent of 16 x 106 cells as previously describe (ref: doi: 10.1371/journal.pgen.1006899. PubMed PMID: 28715449). Immunoprecipitated chromatin was treated with RNaseA. The DNA was isolated using 2% NaSDS and 2mg.ml-1 Proteinase-K, phenol:chloroform extraction using glycogen as carrier and finally ethanol precipitation. ChIP DNA samples were quality controlled by qPCR before being sent for library preparation and 50 or 100 base single-end or paired-end sequencing on an Illumina HiSeq 4000 or NovaSeq 6000 (McGill University and Genome Quebec Innovation Centre).