title: GSM2304719: ESS1.MRE; Homo sapiens; MRE-Seq
library_construction_protocol: DNA extraction used a QIAamp spin column method to yield a mean of 0.68 µg DNA/specimen for LCM sample. For bulk sample, 5-10ug DNA is isolated. For MRE, DNA was isolated using the DNeasy Tissue kit (Qiagen, Valencia, CA). Three parallel digests were performed (HpaII, AciI and Hin6I; Fermentas), each with 1μg of DNA. Five units of enzyme per microgram DNA were added and incubated at 37 °C in Fermentas Tango buffer for 3 h. Digested DNA was precipitated with sodium acetate and ethanol, and 500 ng of each digest were combined into one tube. Combined DNA was size-selected by electrophoresis on a 1% agarose TBE gel. A 100-300 bp gel slice was excised using a sterile scalpel and gel-purified using Qiagen QIAquick columns, eluting in 30 µl of Qiagen EB buffer.Library construction was performed using the Illumina Genomic DNA Sample Kit (Illumina) with single-end adapters, following the manufacturer's instructions with the following changes. For the end repair reaction, T4 DNA polymerase and T4 polynucleotide kinase were excluded and the Klenow DNA polymerase was diluted 1:5 in water and 1 ul used per reaction. For single-end oligo adaptor ligation, adapters were diluted 1:10 in water and 1 ul used per reaction. Fifteen cycles of PCR were performed using the single-end Illumina PCR primers. The resulting reactions were purified with Qiagen MinElute columns, after which a final size selection (220-520bp) was performed by electrophoresis in DNA was eluted in 36 ul EB buffer using Qiagen QIAquick columns. After cleanup with Qiagen MinElute columns, each library was examined by spectrophotometry (Nanodrop, Thermo Scientific) and Agilent DNA Bioanalyzer (Agilent).2% agarose. Libraries were quality controlled by spectrophotometry and Agilent DNA Bioanalyzer analysis.