GSM3454811: MEF MUT; Mus musculus; Bisulfite-Seq
Genomic DNA and RNA were extracted from MEF cultures using AllPrep DNA/RNA/Protein Mini Kit (Qiagen) according to the manufacturer's instructions. Concentration of DNA was measured on Nanodrop and adjusted to 20ng/ul after which the DNA was sheared to 100-500bp fragments on Covaris sonicator with the following settings: 10% duty cycle, 200 bursts/sec, intensity 4.0, 80sec duration; mode – frequency sweeping). Fragment ends were repaired, A-tailed and ligated with PE Illumina methylated adapter oligos using NEBNext DNA Library Prep Master Mix Set for Illumina (NEB). Adaptor-ligated gDNA fragments were subjected to bisulfite conversion using two-step modification procedure with Imprint™ DNA Modification Kit (Sigma). Converted and purified DNA was amplified for 9-12cycles with KAPA HiFi HS Uracil enzyme mix (Anachem). Precise concentration and size of the libraries were determined by qPCR with universal Illumina primers and also on Agilent 2100 Bioanalyzer (Agilent Technologies) using High Sensitivity DNA assay. Libraries were sequenced on HiSeq2000 system (Illumina) using 100bp Paired End protocol. Shared DNA fragment ends were repaired, A-tailed and ligated with PE Illumina methylated adapter oligos using NEBNext DNA Library Prep Master Mix Set for Illumina (NEB). Adaptor-ligated gDNA fragments were subjected to bisulfite conversion using two-step modification procedure with Imprint™ DNA Modification Kit (Sigma). Converted and purified DNA was amplified for 9-12cycles with KAPA HiFi HS Uracil enzyme mix (Anachem). Precise concentration and size of the libraries were determined by qPCR with universal Illumina primers and also on Agilent 2100 Bioanalyzer (Agilent Technologies) using High Sensitivity DNA assay. Libraries were sequenced on HiSeq2000 system (Illumina) using 100bp Paired End protocol.
GEO
GSM3454811