Illumina HiSeq 4000 sequencing; Cooperative binding of DNA sequence non-specific HMGbox Factor Ubf and TBP-TAF complex SL1 determines RNA Polymerase I promoter recognition in vivo and its dysfunction underlies the Ubf-E210K neuroregression syndrome
Cooperative binding of DNA sequence non-specific HMGbox Factor Ubf and TBP-TAF complex SL1 determines RNA Polymerase I promoter recognition in vivo and its dysfunction underlies the Ubf-E210K neuroregression syndrome
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 minor modifications. Briefly, for all cell types, 33 million cells were resuspended in 1.5 ml of Farnham lab buffer (5mM PIPES pH8.0, 85mM 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). The NEXSON-isolated nuclei were recovered by centrifugation (1000g, 5 min, 4째C), resuspended in 1.5 ml of shearing buffer (10mM Tris-HCl pH8.0, 1mM NaEDTA, 0.1% SDS, protease inhibitors) and sonicated for 25 min, 30 sec on : 30 sec off , at high intensity also in the Bioruptor. The resulting chromatin was then cleared by centrifugation (12,000g, 30 min, 4째C). Each immunoprecipitation (IP) was carried out using the equivalent of >16 x 10^6 cells of cleared chromatin fraction as previously describe (ref: doi: 10.1371/journal.pgen.1006899. PubMed PMID: 28715449). Rabbit polyclonal antibodies against mouse and human UBF, RPI large subunit (RPA194/Polr1A), and Rrn3, and against mouse TAF1B were generated in the laboratory (ref: doi: 10.1371/journal.pgen.1006899. PubMed PMID: 28715449) . Other antibodies were obtained commercially: anti-human TAF1B (#D01 Abnova), anti-FLAG M2 (F3165 Sigma). To map PAF53 a cDNA encompassing the complete coding region (NM_022811.3) was isolated from NIH3T3, verified by Sanger sequencing and subcloned into pCDNA3-3xFLAG-C1. The resulting pC3xFLAG-PAF53 (lab. stock #2266) was used to transform NIH3T3 and cells were selected with G418. A pool of positive clones expressing PAF53 at endogenous level was then subjected to parallel ChIP for PAF53 (anti-FLAG), RPI and Rrn3. 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 minor modifications. Briefly, for all cell types, 33 million cells were resuspended in 1.5 ml of Farnham lab buffer (5mM PIPES pH8.0, 85mM 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). The NEXSON-isolated nuclei were recovered by centrifugation (1000g, 5 min, 4째C), resuspended in 1.5 ml of shearing buffer (10mM Tris-HCl pH8.0, 1mM NaEDTA, 0.1% SDS, protease inhibitors) and sonicated for 25 min, 30 sec on : 30 sec off , at high intensity also in the Bioruptor. The resulting chromatin was then cleared by centrifugation (12,000g, 30 min, 4째C). Each immunoprecipitation (IP) was carried out using the equivalent of >16 x 10^6 cells of cleared chromatin fraction as previously describe (ref: doi: 10.1371/journal.pgen.1006899. PubMed PMID: 28715449). Rabbit polyclonal antibodies against mouse and mouse TAF1B were generated in the laboratory (ref: doi: 10.1371/journal.pgen.1006899. PubMed PMID: 28715449). Other antibodies were obtained commercially: anti-FLAG M2 (F3165 Sigma). 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 base single-end sequencing on an Illumina HiSeq 2500 or 4000 (McGill University and Genome Quebec Innovation Centre).
Experimental Factor: genotype
taf1bfl/fl/ER-Cre+/+
Experimental Factor: immunoprecipitate
Ubtf
Experimental Factor: compound
none
Experimental Factor: dose
0