E-MTAB-6271:polstop_red_1_s
NextSeq 500 sequencing; DNA- and RNA-seq to study the impact of 5-formylcytosine on nucleosome organization and gene expression in embryonic mouse tissues and stem cells
DNA- and RNA-seq to study the impact of 5-formylcytosine on nucleosome organization and gene expression in embryonic mouse tissues and stem cells
polstop_red_1_s
Modified synthetic DNA (Widom 601 sequence) was obtained by PCR and used for in vitro nucleosome reconstitution. In vitro assembled nucleosomes were incubated with NaCNBH3 (100mM) in PBS for 16h at 37°C. For polymerase stop assay, reduced nucleosomes using either unmodified or 5fC (100%) modified Widom 601 sequence were obtained. Polymerase extension was achieved in the presence of dNTP (200 µM), primers (0.5 µM), 1 x polymerase buffer and DreamTaq (1 µL, 5U). The mixture was heated at 95 °C for 30 s, 60 °C for 60 s and 72° C for 3 min. After extension, the mixture was treated with Proteinase K (40 µg) in Proteinase K buffer (750 mM Gu-HCl, 5% Tween 20, 30mM EDTA, 30mM Tris-HCl), before purification by Oligo clean and concentrator Kit (Zymo Research). The samples were treated with RecJF (3 µL, 90U) for 12 h at 37 °C to remove excess primer and overhangs. The samples were again purified using the Oligo clean and concentrator Kit before DNA library preparation for sequencing. DNA libraries were obtained by end repair, A-tailing and adapter ligation (NEBNext Ultra II DNA library Prep kit) following the manufacturer's guideline.
Senior research associate
E-MTAB-6271:polstop_red_1_s
Experimental Factor: protocol
DNA-seq
Experimental Factor: organism
synthetic construct
Experimental Factor: genotype
not applicable
Experimental Factor: organism part
not applicable
Experimental Factor: compound
sodium cyanoborohydride
Experimental Factor: immunoprecipitate
none