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== <span style="color:blue">Part II: SeqCap hybridization capture with optimized conditions</span> == * Followed exactly the instruction describe in the User Guide page 22-24 === Step 1. Preparing for hybridization === # Turn on a heat block to 95C. I use thermocycler to heat DNA mix at step 4.9 as I transfer the DNA mix from 1.5mL tube to 0.2ul PCR tube. === Step 2. Resuspend the SeqCap HE Universal and SeqCap HE Index Oligos === * Noi prepared this step the first time she opened the kit * We have HE Oligo Index set A # Spin the lyophilized oligo tubes, contained in the SeqCap HE-Oligo KitA briefly to allow the contents to pellet to the bottom of the tube. # Add 120ul PCR-grade H2O to SeqCap HE Universal Oligo tube (1000uM final concentration). # Add 10ul PCR-grade H2O to SeqCap HE Index Oligo tube (1000uM final concentration). # Vortex the oligos plus H2O for 5s and spin down the resuspended oligo tube. # After use, they should be stored at -15 to -25C === Step 3. Prepare the bisulfite-converted DNA sample library and HE oligos for hybridization === # Have ~1ug of the WGBS library pool in a Lo-bind 1.5 mL tube prepare above, including normal control WGBS library pool and cancer patient WGBS library pool # Thaw the resuspended SeqCap HE Universal Oligos (1000uM) and the resuspended SeqCap HE Index Oligo (1000uM) that matches the DNA adaptor index in the WGBS library preparation ::- In this experiment, I pooled all libraries using barcode adaptor match with all 24 indexes in HE-Oligo KitA/B (0.8ul each). I pool the same amount of the 24 indexes in kit A & B and use 1ul of the pooled oligos in step 4.3 === Step 4. Prepare the hybridization samples === # Add 10ul of Bisulfite Capture Enhancer (found in the ''SeqCap Epi Accessary Kit'') to a new Lo-bind 1.5 mL tube) # Add 1ug of WGBS library pool # Add 1,000pmol SeqCap HE Universal Oligo 1 and 1,000pmol of the 1000uM SeqCap HE Index Oligo SetA as described above. ## This equal to 1ul of 1,000uM of oligos # Close the tube and make a hole in the top of the tube's cap with an 20 gauge needle (I poked many holes) # Dry the mixture of WGBS library pool/Bisulfite Capture Enhancer/Hybridization Enhancing Oligos in a vacuum concentrator on high heat 60C for ~15-20min until it dry up. A little liquid left in the tube between 1-2ul should be fine to prevent over dry. # Cover the holes in the tube's cap with a sticker # To each tube, add ## 7.5ul of 2X Hybridization Buffer (vial 5) ## 3ul of Hybridization Component A (vial 6) # Vortext the mixture for 10s and centrifuge at maximum speed for 10s. I transfer the contents in the tube to 0.2mL Lo-bind PCR tube. Make sure that you resuspend the mixture very well. # Heat the mixture at 95C for 10min with a heat lid on thermocycler. During this time, I thaw a 4.5ul aliquot of SeqCap Epi probe pool in a 0.2mL PCR tube on ice and spin down # Centrifuge the heated tubes at maximum speed for 10s at RT. # Transfer the WGBS library pool/Bisulfite Capture Enhancer/Hybridization Enhancing Oligos/Hybridization Cocktail to the 4.5uL SeqCap Epi probe pool on ice. # Vortex quickly for 3s and at maximum speed for 10s at RT. # Add 20l mineral oil on top. # Incubate on a thermo cycler at 47C for 64-72h. Turn on the thermocycler 's heat lid and set to maintain at 57C (10C above the hybridization temperature). * NOTE: I overlaid the hybridization sample with 20ul mineral oil as Alice did before to prevent evaporation '''The hybridization sample contains the following components:''' '''Component''' '''Solution Capture''' Bisulfite Capture Enhancer 10ug WGBS libraries 1ug Hybridization Enhancing Oligos 2,000pmol* ------------------------------------------------------- (All component above were heated up to dry) 2X Hybridization Buffer (vial 5) 7.50ul Hybridization Component A (vial 6) 3.00ul SeqCap Epi probe pool 4.50ul '''Total''' '''15.00ul''' * Composed of 50% (1,000pmol) SeqCap HE Universal Oligo & 50% (1,000pmol) of the appropriate SeqCap HE Index Oligo. * For this experiment, I incubate the hybridization sample for 77h and transferred the reaction to 4C to continue the next day.
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