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===Preparation of spiked-in lambda DNA (protocols taken direct from publication) === *'''IMPORTANT: We need to design & make probes to capture the spiked-in sequences''': We need to make sure that the samples will have spiked-in controls (both control A for the methylC-Seq and control B for TAB-seq). *'''Generation of Spiked-in Conversion Controls for H1 Cells''': Several spiked-in controls were generated and tested. Spike-in control A consisted of a 1:1 mixture of unmethylated lambda DNA (Promega Cat. No. D1521) with M.SssI-converted pUC19 DNA (NEB, Cat. No. M0226S). To generate the spiked-in control B, unmethylated lambda DNA (Promega Cat. No. D1521) was PCR amplified and purified by gel electrophoresis in non-overlapping 2 kb amplicons, with a cocktail of dATP/dGTP/dTTP and either: d5mCTP (Zymo Research, Cat. No. D1035) at genomic positions 0β10 kb, dCTP at genomic positions 20β30 kb, and d5hmCTP (Zymo Research, Cat. No. D1045) at genomic positions 38β48 kb. Amplicons with d5mCTP/d5hmCTP and dCTP were amplified by ZymoTaq DNA polymerase (Zymo Research, Cat. No. E2001) and Phusion HF DNA polymerase (NEB, Cat. No. M0530S), respectively, as per manufacturers' instructions. Spiked-in DNA was added to H1 genomic DNA to a final concentration of 0.5% (control A for replicate 1, control B for replicate 2), and sonicated to a range of 300β500 bp with a Biorupter 300 (high power, 15 s on, 15 s off, 20 cycles).
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