GSM1619028: SW48 DNA methylation; Homo sapiens; OTHER
phenol-chloroform Five micrograms of genomic DNA spiked with 500 ng of methylation standards with defined methylation levels of 0, 25, 50, and 100% were digested with 100 units of SmaI endonuclease (NEB) for 3 hours at 25°C. Subsequently, 100 units of XmaI endonuclease (NEB) were added and the digestion was continued for an additional 16 hours at 37°C. Digested DNA was purified using the QIAquick PCR purification kit (Qiagen, Valencia, CA) and eluted in TRIS-HCl 10 mM pH 8.5 (EB). Eluted DNA was supplemented with NEB buffer #2, dCTP, dGTP and dATP (0.4 mM final concentration of each), 15 units of Klenow Fragment (3'→5' exonuclease deficient) DNA polymerase (NEB) and incubated for 30 minutes at 37°C. This step filled in the recesses at 3' DNA ends created by XmaI digestion and added 3' dA tails to all fragments. 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).
GEO
GSM1619028
GEO Accession
GSM1619028