title: GSM3937914: 601_S1S2_RSC_RAP1; synthetic construct; MNase-Seq
library_construction_protocol: RSC sliding reactions were performed in reaction buffer (10mM Tris pH7.4, 150mM KCl, 3mM MgCl2, 0.1mg/ml BSA) and a total volume of 70µl. The following components were added in sequential order mononucleosomes (to make 20nM final concentration), Nap1 (10 Nap1 : 1 mononucleosome ratio), Rap1 (10 Rap1 : 1 mononucleosome ratio, for w/o Rap1 MQ water was used as substitute), RSC complex (1 RSC : 5 mononucleosome ratio) and finally ATP (1mM). Reactions were placed at 30°C for 90 mins after which 10ul was taken and glucose was added to make 8% final concentration and loaded onto commercial Criterion Precast Gel (Biorad) 5% acrylamide, 1mm, run in 1xTBE at 200V for 35-45mins on ice. Gels were stained in Gelred and imaged using ChemiDoc MP (Biorad). To the remaining 60ul, 60ul 50mM Tris-HCl pH 8 and 10x NEB MNase buffer (M0247S) (to make final 1x) was added. This 120ul total sample was split into 3 x 40ul aliquots and to each either 6 units, 3 units or 1 unit of Mnase (M0247S) was added respectively and left to digest for 5 mins at 37° C. To stop the reaction an equal volume of stop buffer was added (200mM NaCl, 30mM EDTA, 1% SDS) and left on ice for 5 mins. Finally, 10µg of Proteinase K (Sigma P2308) was added and left for 1h at 60° C and DNA fragments were isolated using QIAquick PCR purification spin columns (Qiagen). For nucleosome only samples (t0), reactions were performed directly in 1x NEB Mnase buffer (M0247S), Mnase and Proteinase K digestion as well as DNA fragment purification was performed as described for RSC assay nucleosomes. All purified samples, were run on a 2% agarose 1xTBE gels for run in 1xTBE at 110V for 60min. Gels were stained in Gelred and imaged using ChemiDoc MP (Biorad). TruSeq ChIP library preparation kit (Illumina)
attributes.tag: GEO Accession
attributes.value: GSM3937914