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_control_1_s
library_construction_protocol: Modified synthetic DNA (Widom 601 sequence) was obtained by PCR and used for in vitro nucleosome reconstitution. 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: none
attributes.tag: Experimental Factor: immunoprecipitate
attributes.value: none