title: Illumina HiSeq 2500 sequencing; Identification of the primary target of the chemotherapeutic agent CX-5461 as a late step in transcription initiation by RNA Polymerase I
design: Identification of the primary target of the chemotherapeutic agent CX-5461 as a late step in transcription initiation by RNA Polymerase I
library_construction_protocol: 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, 4°C), 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, 4°C). 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. NIH3T3 and HEK293T were cultured in DMEM, 10% FBS as described by American Type Culture Collection (ATCC). 10mM stock of CX-5461 (Calbiochem/EMD Biosciences) was prepared in 50mM NaH2PO4 (vehicle). The CX-5461 or vehicle were then diluted in culture medium to obtain the required final concentration. Actinomycin D (Calbiochem/EMD Biosciences) was prepared in DMSO at 5mg/ml and this or an equivalent volume of DMSO diluted in culture medium to obtain a final concentration of 50ng/ml (40nM). Treatment was terminated by cell fixation for ChIP. 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).