title: WGBS of Mus musculus: NHEJ1_zr2353_1 male spleen
design: Initially, 5 mg of excised spleen tissue was mechanically disrupted using liquid nitrogen grinding.  Genomic (gDNA) was isolated using a modified protocol of the Genomic DNA Isolation Kit (AbCam; #ab65358).  Using 35 L of cell lysis buffer, the pulverized tissue was washed by centrifugation.  The nuclei were suspended in 40 L of cell lysis buffer with 5 uL of kit enzyme mix, mixed by gentle pipetting, and incubated in a 55 C water bath for 1 h.  gDNA was isolated from the extraction mixture using a modified protocol of the paramagnetic bead DNA isolation in the ChargeSwitch gDNA Micro Tissue Kit (Thermo Fisher Scientific; #CS11203).  1 ml ChargeSwitch Lysis Buffer (L15) and 10 L of proteinase K were added to the nuclei lysate.  The mixture was incubated in a 55 C water bath for 1 h.  After incubation, 5 L of RNase A was added to the lysate and homogenized by pipetting.  The mixture was incubated at room temperature for 5 min.  gDNA was isolated following the DNA binding, DNA washing, and DNA elution processes described by the manufacturer protocol.  The elution volume was concentrated using a Savant DNA120 SpeedVac Concentrator (Thermo Fisher Scientific) to a volume of 100 L.  The concentration of recovered gDNA for each sample was measured using the Qubit 3.0 fluorometer system (Thermo Fisher Scientific).  Quality of each sample was evaluated using a DNA 12000 Chip Kit (Agilent; #5067-1508) on a 2100 Bioanalyzer System (Agilent; #G2939BA).  Bisulfite conversion and sequencing was performed by Zymo Research, using a Methyl MaxiSeq service.  Briefly, methyl-seq libraries were prepared using 500 ng of genomic DNA, fragmented with 2 units of dsDNA Shearase Plus (Zymo Research; #E2018-50).  The resulting genomic fragments were end-blunted and the 3 terminus was modified with an adenine extension.  The fragments were subsequently purified using the DNA Clean & Concentrator-5 kit (Zymo Research; #D4003).  A-tailed genomic fragments were ligated to pre-annealed adapters containing 5-methylcytosine in place of cytosine.  Adapter-ligated fragments were repaired to complete duplex strands.  Bisulfite conversion of the fragments was performed using the EZ DNA MethylationLightning kit (Zymo Research; #D5030).  The fragments were amplified by polymerase chain reaction using adapter targeted primers with Illumina TruSeq indices.  The size and concentration of the fragments were confirmed using an Agilent 2200 TapeStation.  Methylome fragments were sequenced on an Illumina HiSeq 1500 instrument, generating paired-end 150-bp read lengths.