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== Preparation of genomic DNA for BS-seq and oxBS-seq == * While the Nature Protocol reference states that DNA preparation for targeted sequencing does not need to be sonicated, the TrueMethyl application note protocol 03 recommends Covaris shearing the genomic DNA to 10 kbp before moving on. === Needle shearing method instead of Covaris shearing === * Cheap way to shear the DNA * [[File:smrtcommunity_needle_shearing_25kbp.pdf]] * UV the syringe needle * Passing a solution of 1-10 ug of genomic DNA through a 26 gauge syringe needle can also shear the DNA. ** Pass at least 5 times to get 25 kbp fragments. Can run a gel to check. === Shear DNA with QIAEXII beads=== * Covaris tubes didn't shear well, decided to use QIAEXII beads instead. * Follow beads purification and add vigorous vortexing for 90s. === Add digestion control to the DNA to achieve 1% wt/wt.=== * Use 1.6X AMPure XP beads to purify and concentrate the DN. Wash the beads with 80% ethanol twice and let air dry for 30 minutes after the last wash. Elute the DNA with 22.5 uL of nuclease-free water. * Prepare a BioRad Micro Bio-Spin P-6 (preferably SSC buffer) for preoxidation purification. ** centrifuge tube at 1000 g for 120 s ** wash 3 times with 500 uL water at 1000 g and 60 s ** wash 1 last time with 500 uL water at 1000 g and 120 s ** Add DNA sample to the column, spin at 1000 g for 240 s. Retain the eluate.
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