title: Illumina NovaSeq 6000 paired end 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
design: 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
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 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). To map Ubtf1 and Ubtf2 variants cDNAs encompassing the complete coding regions (Genbank: M61726 / M61725) were subcloned into pCDNA3-3xFLAG-C1 (N. Bisson) and verified by Sanger sequencing. The resulting pC3xFLAG-UBF1 and -UBF2 (lab. stocks #2072, #2073) constructs were used to transfect NIH3T3 or conditional ubtf (ubtffl/fl/ER-Cre+/+/p53-/-) MEFs (Herdman et al., 2017) and cultures selected with G418. In the case of NIH3T3, pools of positive clones expressing Ubtf1 or Ubtf2 at sub-endogenous levels were then subjected to parallel ChIP for FLAG-Ubtf1/2 (anti-FLAG) and total Ubtf. Essentially the same procedure was used for the MEFs except that cells were transfected with pCDNA3Hygro-3xFLAG-UBF1 and -UBF2 (lab. stocks #2273, #2274), expressing clones selected with Hygromycin and these used in ChIP experiments 5 days after ubtf inactivation by 4-HT addition or after mock inactivation to eliminate endogenous Ubtf.  DNase-Seq analysis of MEFs was carried out following the published protocol (ref: doi: 10.1038/nmeth.2762. PubMed PMID: 24317252), the only modifications being adjustment of digestion levels to obtain the required fragmentation. DNA samples were quality controlled by qPCR before size selection following the published protocol. 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 or paired-end sequencing on an Illumina HiSeq 2500 or 4000, or NovaSeq 6000 (McGill University and Genome Quebec Innovation Centre).