GSM3400991: DNA_METH_RISP_KO2; Mus musculus; Bisulfite-Seq
Genomic DNA was isolated using the AllPrep DNA/RNA Micro Kit (Qiagen). Genomic DNA was quantified with a Qubit 3.0 instrument. ~50-200 ng of genomic DNA was then digested with the restriction endonuclease MspI (New England BioLabs). Resulting fragments underwent size selection for fragments ~100-250-bp in length using SPRI beads (MagBio Genomics) and subsequent bisulfite conversion using the EZ DNA Methylation-Lightning Kit (Zymo Research) per the manufacturer's protocol. Bisulfite conversion efficiency averaged 99.4% as estimated by the measured percent of unmethylated CpGs in λ-bacteriophage DNA (New England BioLabs N3013S) added at a 1:200 ratio to each sample. Libraries for Illumina-based sequencing were prepared with the Pico Methyl-Seq Library Prep Kit (Zymo Research) using Illumina TruSeq DNA methylation barcodes. Libraries were run on a High Sensitivity chip using an Agilent TapeStation 4200 to assess size distribution and overall quality of the amplified library. Fluorometric quantitation and TapeStation size distribution estimates permitted equimolar pooling, and 4-6 pooled libraries per run were sequenced on an Illumina NextSeq 500 instrument using the NextSeq 500/550 V2 High Output reagent kit (1 x 75 cycles).
GEO Accession
GSM3400991