title: GSM2401522: Mnase-seq Control_t4_R2; Saccharomyces cerevisiae; MNase-Seq
library_construction_protocol: Cross-linked chromatin–DNA complexes were extracted by mechanical disruption (bead beating; 4 1 min sessions, 2 min on ice between sessions).  An added level of standardization was achieved by quantifying the input of extracted chromatin loaded into each digest reaction; 1 mg total protein from whole-cell extracts per 200 ml digest reaction. Total protein concentration for whole cell extracts (chromatin extracts) was quantified using a Bradford assay (OD595). Following MNase treatment and cross-link reversal, DNA was phenol: chloroform precipitated, resuspended in 50 ml TE pH 8.0, and RNase treated. Small aliquots (20%,  10 ml) from each digest titration were then analyzed by gel electrophoresis. Gel intensity measurements for each lane were calculated using standard densitometry software provided by Biorad (Quantity OneTM) and exported to Microsoft ExcelTM for correlation analysis (Pearson). Correlation coefficients were calculated for digest titration levels no longer showing a visible di-nucleosome band (i.e. optimally digested samples) and spanning a region known to cover a size range of 0–400 bp of DNA. A 100-bp standard ladder (Qiagen) was analyzed on the same gel to calculate a relative front (i.e. separation relative to standard fragment sizes) for the migrating DNA population. ‘Matched’ digests were selected as two optimally digested samples having a correlation coefficient (r>0.9). Correlation coefficients r>0.9 were selected because these showed the most reproducible change in MNase protection data on single site real-time quantitative PCR (RT-qPCR) analysis (See Supplementary Methods section). The remaining sample from each optimal digest was column purified (Zymo Research), bypassing the need to gel excise mono-nucleosome DNA. Avoidance of gel extraction to isolate chromatin DNA at this stage is a key step to assure sampling of similar chromatin populations. Matched DNA samples were prepared for sequencing using the standard Illumina protocol with additional care at the gel purification step to ensure that the same size range of DNA was selected.
identifiers.namespace: GEO
identifiers.id: GSM2401522