GSM4708553: TAPSbeta; Mus musculus; Bisulfite-Seq
The genomic DNA was prepared by cell harvesting with centrifugation for 5 min at 1000 x g and room temperature, and DNA extraction with Quick-DNA Plus kit (Zymo Research) according to the manufacturer's protocol. mESC gDNA was spiked with 0.5% of methylated lambda DNA, 0.025% of 2kb-unmodified and 0.025% of 2kb-caC spike-in controls. For CAPS approach, gDNA was fragmented by Covaris M220 instrument and size-selected to 200-400 bp using Ampure XP beads (Beckman Coulter). For other approaches, gDNA was fragmented and size-selected to 300-500 bp. 0.01% of synthetic oligo with N5mCNN /N5hmCNN sequences and 0.01% of synthetic oligo with 5fC modifications were added after size-selection. 100 ng of fragmented DNA was used for end-repair/A-tailing and ligation of NEBNext Adaptor (NEB) with KAPA Hyper kit (KAPA) according to the manufacturer's protocol. The uracil in the loop of NEBNext Adaptor was removed by adding 3 μL of USER enzyme (NEB) to the ligation reaction and incubating for 15 min at 37°C. Then the reaction was purified with 0.8X Ampure XP beads according to the manufacturer's protocol. For CAPS approach, 80% of acetonitrile: H2O was used instead of 80% ethanol: H2O during beads purification step. OTHER: subtraction-free and bisulfite-free specific sequencing of DNA methylation
GEO Accession
GSM4708553