GSM3937912: 601_S1S2; synthetic construct; MNase-Seq
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)
GEO Accession
GSM3937912