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==Protocol== ===TagmentationTM Reaction === *1. Prior to assembling the reaction, briefly centrifuge ALL tubes to assure that the reagents are at the bottom of the tubes. *2. Assemble the following reaction components on ice, in the order listed: 12.67ul Nuclease-Free Water 2.33ul 100ng NA12878 gDNA (42.9ng/ul nanodrop check on 2/15/11) 4ul 5X Nextera HMW Reaction Buffer 1ul Nextera Methyl-Seq Tagmentation Mix (Illumina-compatible) 20ul Total reaction volume *3. Mix briefly by vortexing, and incubate at 55C for 5 minutes. *4. Purify the tagmented DNA using Qigen column and elute with 11ul Elute buffer *5. Add 10 μl of 2X Nextera Fill-in Buffer and 1 μl of Nextera Methyl-Seq Polymerase to 10 μl of the eluted/Tagmented DNA. Mix and incubate at 70C for 10 minutes. *6. Purify the Fill-in reaction using a Qiagen column as in Step 4 above. ===Bisulfite Conversion of Tagged Nextera Fragments=== *1. Add 900ul water, 300ul of M-Dilution Buffer, and 50ul M-Dissolving buffer to a tube of CT Conversion Reagent *2. Mix at room temperature with frequent vortexing or shaking for 10 minutes *3. Preparation of M-Wash buffer *4. Mix 10ul nuclease free water with 130ul of the CT Conversion Reagent to 10ul of your DNA sample in a PCR tube *5. Place the sample tube in a thermal cycler and perform the following steps 98C for 10mins 64C for 2.5hrs 4C forever *6. Add 600ul M-binding buffer to a Zymo spin column and place the column into a provided collection tube *7. Load the sample into the column. Close the cap and mix by inverting the column several times *8. Centrifuge at full speed for 30sec. Discard the flow through *9. Add 100ul M-wash buffer to the column. Centrifuge at full speed for 30sec *10. Add 200ul M-Desulphonation Buffer to the column and let stand at room temperature for 20mins. After incubation centrifuge at full speed for 30sec *11. Add 200ul M-Wash buffer to the column. Centrfuge at full speed for 30sec. Add another 200ul of M-wash buffer and centrifuge for an additional 30sec *12. Place the column into a 1.5ml microcentrifuge tube . Add 15ul of M-Elution Buffer directly to the column matrix. Centrifuge for 30sec at full speed to elute the DNA ===Addition of bPCR-Compatible Sites and Library Enrichment=== *1. Assemble the following reaction components at room temperature 12ul Nuclease-Free water 10ul bilsufite converted library 25ul 2X Nextera PCR Buffer 1ul 50X Nextera Primer Cocktail 1ul 50X Nextera Adaptor 2 1ul Nextera MEthyl-Seq Polymerase *2. Cycle the samples in a thermocycler under the following conditions 95C for 30sec 25cycles of 95C for 10sec 60C for 10sec 72C for 1min hold for 1min *3. Purify the tagged DNA using qiaquick column
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