organism: synthetic construct
description: Protocols: 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: ENA first public
attributes.value: 2018-10-22
attributes.tag: ENA last update
attributes.value: 2017-12-07
attributes.tag: External Id
attributes.value: SAMEA104420740
attributes.tag: INSDC center alias
attributes.value: Senior research associate
attributes.tag: INSDC center name
attributes.value: Senior research associate
attributes.tag: INSDC first public
attributes.value: 2018-10-22T17:03:09Z
attributes.tag: INSDC last update
attributes.value: 2017-12-07T13:57:46Z
attributes.tag: INSDC status
attributes.value: public
attributes.tag: Submitter Id
attributes.value: E-MTAB-6271:polstop_red_1
attributes.tag: broker name
attributes.value: ArrayExpress
attributes.tag: sample name
attributes.value: E-MTAB-6271:polstop_red_1