title: GSM2458790: Empty Vector_1 Dream; Homo sapiens; OTHER
library_construction_protocol: DNA isolation: The pellet was lysed in the solution containing 2% sodium dodecyl sulphate and 25 mM EDTA. Proteins were precipitated by adding 1/3 volume of 10 M ammonium acetate and removed as a pellet by centrifugation. DNA was precipitated from the supernatant with isopropanol, washed with 70% ethanol and dissolved in TE (10 mM TRIS pH 8.0, 1 mM EDTA). Two micrograms of genomic DNA spiked with 5 methylation standards with defined methylation levels were digested with 20 units of SmaI endonuclease (NEB) for 8 hours at 25°C. Subsequently, 20 units of XmaI endonuclease (NEB) were added and the digestion was continued for an additional 16 hours at 37°C. Digested DNA with methylation-specific signatures at the ends of restriction fragments was end repaired using dCTP, dGTP and dATP (0.4 mM final concentration of each) and 15 units of Klenow Fragment (3'>5' exonuclease deficient) DNA polymerase (NEB). Illumina paired end (Quail et al. 2008) or barcoded Truseq (Illumina, San Diego, CA) sequencing adapters were then ligated at 10:1 adapter:fragment ratio using Rapid T4 DNA ligase (Enzymatics, Beverly, MA). The ligation mix was size selected by electrophoresis in 2% agarose. Two slices corresponding to 250-350 bp and 350-500 bp sizes based on a 100 bp DNA ladder (NEB) were cut out and DNA was extracted from agarose. DNA eluted from the slices was separately amplified with Illumina paired end PCR primers (Quail et al. 2008) using iProof high-fidelity DNA polymerase (Bio-Rad Laboratories, Hercules, CA) and 18 cycles of amplification. Resulting sequencing libraries were purified with AMPure magnetic beads (Agencourt, Beverly, MA).
attributes.tag: GEO Accession
attributes.value: GSM2458790