GSM2474320: RNA-seq of Arabidopsis thaliana: Dark Grown [Dark-RNA3]; Arabidopsis thaliana; RNA-Seq
Chromatin Digestion Pelleted cell culture was ground in a liquid nitrogen-cooled pestle and mortar to generate a cell powder. 1mL of the powder was suspended in a 0.5mL modified spheroplast digestion buffer & Nonidet P40 (SDBN: 1M sorbitol, 10mM NaCl, 50 mM TrisŸHCl pH7.5, 5mM MgCl2, 1 mM CaCl2, 1 mM 2-mercaptoethanol, 0.5 mM spermidine, 0.075% Nonidet P40). 300 µl of cells were transferred into 1.5mL microcentrifuge tube containing 30 or 120 units of MNase (Affimetrix). Cells were digested with MNase at 37°C for 3 min. The MNase digestion reactions were stopped by addition of and thorough mixing 30 µl of STOP solution containing 5% SDS and 250 µl EDTA. DNA was extracted by addition of an equal volume of 2X CTAB supplemented with PVP (200 mM Tris-HCl, 40 mM EDTA, pH 8.0; 2.8 M NaCl, 4% w/v CTAB, 2% PVP-40 cas number 9003-39-8) was added to the digestion reaction and incubated at 45°C for 15 mins. DNA was separated from protein by two phenol:chloroform (300:300) steps. DNA was precipitated with sodium acetate and propan-2-ol then washed in 80% ethanol and dried. Samples were incubated with 10X RNase A at 37°C for 1h with 100U unmodified T4 polynucleotide kinase (NEB) for further 30 min at 37°C. MNase digested DNA was separated on 1.5% agarose gel, stained with ethidium bromide for 10 min at 80V, DNA fragments ranging from 20 bp to 1 kb were cut out and gel pieces containing DNA were placed in a Costar Spin-X 0.45 µm cellulose acetate centrifuge tube filter (Sigma CLS8136). Two series of freeze-thaws (-80C for 10 mins/RT for 10 mins) were performed to macerate the gel. Filter tubes were centrifuged twice (14kg; 5 min; RT) with a 180 degree rotation of the tubes between the two spins. DNA in the aqueous phase was then extracted with phenol:chloroform (200:200), and precipitated at -80°C for 30 mins with sodium acetate to propan-2-ol and finally washed with 80% ethanol before being resuspended in  ultrapure milliQ water. RNA isolation Total RNA was extracted using the RNeasy Plant mini Kit (Qiagen, http://www.qiagen.com). Nucleic acid quantification and quality check DNA samples were quantified on QubitTM 2.0 Fluorometer with dsDNA BR assay according to manufacturer’s instructions. RNA was quantified using QubitTM 2.0 fluorometer with Qubit RNA BR assay according to manufacturer’s instructions. RNA quality was checked on 1.5% agarose gel. Exeter Sequencing Service and Computational core facilities at the University of Exeter performed the Genomic and RNA library preparation, quantification and 50bp paired end sequencing on HiSeq 2500.
GEO Accession
GSM2474320