title: 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
design: DNA- and RNA-seq to study the impact of 5-formylcytosine on nucleosome organization and gene expression in embryonic mouse tissues and stem cells
library_name: polstop_red_1_s
library_construction_protocol: 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.
attributes.tag: Experimental Factor: protocol
attributes.value: DNA-seq
attributes.tag: Experimental Factor: organism
attributes.value: synthetic construct
attributes.tag: Experimental Factor: genotype
attributes.value: not applicable
attributes.tag: Experimental Factor: organism part
attributes.value: not applicable
attributes.tag: Experimental Factor: compound
attributes.value: sodium cyanoborohydride
attributes.tag: Experimental Factor: immunoprecipitate
attributes.value: none