GSM2407051: GFP DNA rep1; Drosophila melanogaster; MNase-Seq
Chromatin (DNA) preparation: Cells were washed twice with 1X PBS, then gently permeabilized using 950µl spheroplast digestion buffer containing NP-40 (SDBN, 1M Sorbitol, 50mM NaCl, 10 mM Tris-HCL [pH7.5], 5mM MgCl2, 1mM CaCl2, 0.5mM Spermidine, 1mM β-Mercaptoethanol, 0.075% NP-40). Micrococcal Nuclease was added to a final concentration of 600U/ml, and the plate was gently rocked for 3 minutes at room temperature. The reaction was stopped with 100µl STOP solution (5% SDS, 250mM EDTA). DNA was extracted using 2 phenol/chloroform purifications, with RNase treatment (5µl, 10mg/ml, 30 minutes, 37oC) after the first purification. 100% ethanol containing 0.3M NaOAc was used to precipitate DNA. RNA: Cells were harvested from 1x4cm area of plate after PBS washes (see chromatin protocol) but before digestion. Total RNA was harvested using the RNAeasy mini kit (Qiagen) as per manufacturer's guidelines. NKD DNA: The genomic DNA extracted from a single fly was incubated with 22u/ml Mnase for 20 seconds.  The reaction was stopped with 100µl STOP solution (5% SDS, 250mM EDTA).  RNase treatment (5µl, 10mg/ml, 30 minutes, 37oC) was followed with DNA extraction using a phenol/chloroform purification. 100% ethanol containing 0.3M NaOAc was used to precipitate DNA. DNA: All sequencing performed by Exeter University Sequencing service. Standard Illumina protocols were performed for samples being sequenced on a Illumina HiSeq 2000 machine running in paired-end 100bp mode. During size selection, primer dimers were removed and all other DNAs were used for sequencing. RNA: All sequencing performed by Exeter University Sequencing service. Standard Illumina protocols were performed for samples being sequenced on a Illumina HiSeq 2000 machine running in paired-end 100bp mode. RNA was run through an oligo dT column prior to cDNA generation to filter for mRNA transcripts only
GEO Accession
GSM2407051