GSM5456793: HPVneg11; Homo sapiens; Bisulfite-Seq
Samples were quantitated using Qubit, and quality was assessed on the TapeStation 2200 with the genomic DNA tapescreen kit. 300ng of gDNA was sheared with a Covaris S220 (Duty factor 5%, Peak incident 105, Cycle/burst 200, time 80s, temperature 4-8C) in a 130ul volume. Sheared DNA was cleaned using Qiagen PCR Purification columns, with an elution volume of 32ul EB. Sheared DNA was end-repaired, A-tailed, and ligated with Illumina Methylation adapters overnight at 16C. The enzyme was heat inactivated at 65C for 10 min before clean up of DNA using Qiagen MinElute PCR Purification columns and elution in 20ul EB. Ligated DNA samples ran on a 1% agarose gel to select fragments 200-650bp and clean up with Qiagen Gel Extraction kit. The samples were next processed for bisulfite treatment using Zymo's EZ DNA Methylation kit. The reactions were prepared according to the manufacturer's instructions, but incubated in a thermocycler as follows: 55 cycles of 95C for 30sec, 50C for 15 min. After cleanup of the DNA from bisulfite reaction, the libraries were amplified using Roche FastStart Hi-Fidelity DNA polymerase, and PCR PE primers for 16 cycles. The PCR products were cleaned using Agencourt AMPure XP bead 1.8:1 bead:DNA ratio. Final libraries were quantified with Qubit BR dsDNA kit, and the library size was assessed on a TapeStation 2200 using a HS HD1000 screentape kit.
GEO
GSM5456793