title: GSM2935179: T162A [MeDIP-seq]; Homo sapiens; MeDIP-Seq
library_construction_protocol: 1.5µg of original genomic DNA was fragmented to DNA smear between 200bp to 500bp using a Bioruptor NGS (Diagenode) sonication system. End repair, <A> base addition and adaptor ligation steps were performed using Illumina's Paired-End DNA Sample Prep kit following the manufacturer's instructions. Then, the methylated DNA fractions were immunoprecipitated using the Magnetic Methylated DNA Immunoprecipitation Kit (Diagenode, catalog number: mc-magme-048) with small adjustments. Briefly, 1.5µl of each two control templates (methylated and unmethylated DNA controls) with different sequences were mixed with adapter ligated DNA and heat denatured (95°C, 3min) in a final reaction volume of 90µl within the provided reagents of 24µl MagBuffer A and 6µl MagBuffer B. 7.5µl aliquot of the denatured genomic DNA was saved as input and another 75µl was immunoprecipitated with 5µl of prepared anti-5mC antibody and 20µl of prepared Magbeads. The DNA-antibody-magbeads mixture was incubated overnight at 4°C on a rotating wheel. The DNA-antibody-magbeads mixture was then washed twice using each of 150µl ice-cold MagWash Buffer-1 and washed once with 150µl of ice-cold MagWash Buffer-2. For each buffer, the washing reaction was incubated for 4min at 4°C on a rotating wheel. The input DNA and immunoprecipitated products were then in parallel treated with protease K and purified with ZYMO DNA Clean & Concentrator-5 (ZYMO). The efficiency and sensitivity of immunoprecipitation reaction were detected by qPCR using the enriched DNA and the unbound input DNA. MeDIP-seq library was constructed by PCR amplification with the enriched DNA as template. PCR reaction was performed in 50µl of reaction volume consisting of 20µl MeDIP enriched DNA, 5µl of 2.5mM dNTP, 2µl primers, 5µl of 10X pfx Amplification Buffer, 2µl of 50mM MgSO4, 15.2µl sterilized water and 0.8µl of Platinum pfx DNA polymerase (Invitrogen). The program of amplification was 94°C 2 min, 10 cycles of 94 °C 15s, 62°C 30s and 72°C 30 then prolong with 10min at 72°C. The products could be kept at 12°C. The PCR products were purified using Agencourt Ampure Beads (Beckman Counlter). After analyzing by the Bioanalyzer analysis system (Agilent, Santa Clara, USA) and quantified by the real time PCR, the prepared library was sequenced using Illumina HiSeq2000 with SE50 read length. DNA libraries were prepared for sequencing using standard Illumina protocols
attributes.tag: GEO Accession
attributes.value: GSM2935179