title: GSM2055777: MIC_heavy_MNase_rep1; Tetrahymena thermophila CU428; MNase-Seq
library_construction_protocol: Mononucleosomal DNA was purified using two methods: agarose gel electrophoresis and sucrose gradient.     For  gel electrophoresis, Add 10 mM EGTA, 1 mM EDTA and 1 % [v/v] SDS and sit at RT for 5 min to stop reaction. DNA was purified through phenol-chloroform extraction after RNAse A and ProK treatment, then ethanol-precipitated, and resuspended in ddH2O. 2 ul of DNA from each sample was run on a 2 % agarose-TAE gel to check the progression of MNase digestion. Mononucleosomal DNA was subsequently purified by Gel DNA recovery kit (Zymoclean) after collecting the proper agarose gel band;     For  sucrose gradient, Add 10 mM EGTA, 1 mM EDTA and 5 mM Na Butyrate to stop reaction. Add 0.1 % [v/v] TX-100 to do nucleus permeabilization and sit on ice for 10 min. Digested DNA with histone was cleaned by centrifugation at 3000 g, 4 oC for 10 min. Mononucleosome was collected by centrifugation at 103000 g, 4 oC for 17 hours in sucrose gradient buffer (10 mM Tris-HCl, 0.1 mM EDTA, 0.1 M NaCl, 5 mM Na Butyrate, with 5%, 10%, 15%, 20%, 25% sucrose, in total 5 sucrose gradient layer, 1.2ml for each layer). Use loading tips (200 ul) to take supernatant, from top to bottom, 200 ul each time to wells of UV-compatible 96-well plate carefully. Measure OD260 & OD280 of each well. According to the light absorption curve, proper fractions containing mononucleosome were collected. Coomassie brilliant blue staining was performed to validate the presence of histones. DNA was subsequently purified through phenol-chloroform extraction after RNAse A and ProK treatment, then ethanol-precipitated, and resuspended in ddH2O. 2 ul of DNA from each sample was run on a 2 % agarose-TAE gel to check the progression of MNase digestion. Purified nucleosome–sized DNA after MNase digestion was used to prepare Illumina sequencing libraries according to manufacturer’s instructions without the fragmentation step. Paired-end sequencing was peformed using Illumina platform.
attributes.tag: GEO Accession
attributes.value: GSM2055777