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=8-CVI, 9-CVI, 8-CVF, 9-CVF, 8-NA, 9-NA, 8-DF,8-FS, 9-DF, 9-FS, 8-PGP1-iPS= *Fragmentation and end-polishing *Size Selection *Ligation *PCR of sequencing Library *QPCR quantification ===Fragmentation and end-polishing (Make blunt ends with 5'P)=== *The PCR amplicons (200ng-1ug) are fragmented with Covaris sonicator to ~100bp *End-Repair Reactions {| {{table}} | Fragmented DNA|| 85ul |- | 10X End Repair Bufer|| 10ul |- | End Repair Enzyme Mix|| 5ul |- | |} *Incubate tubes at RT for 30minutes. *Perform a Qiaquick purification and elute with 39ul EB buffer. *NOTE: For blunt-end ligation, the A-Tailing reaction should be skipped. Doing TA ligation is preferable since it eliminates the chance of getting chimeric reads. *A-Tailing Reactions {| {{table}} | Blunet-end DNA||37ul |- | 10X dA-Tailing Reaction Buffer||5ul |- | Klenow Fragment (3'-5' exo-)||3ul |- | H2O||5ul |- | |} ===Size selection using Invitrogen 2% SizeSelect gel=== *Fill any unused well with 30ul EB Buffer *Mix 15ul EB buffer with 0.5ul ladder in a 0.2ml tube *Load 20u end-polished DNA sample into two lanes(use 3 or more lanes if the starting DNA is more than 1ug) *Run the SizeSelect program for 12~14 min. Pause when the 125bp band just move into the middle collection well *Use pipette to extract all solution in each of the sample well, and quickly refill the wells with 20ul EB. *Resume the electrophoresis for additional 15sec, extract DNA and refill the wells with 20ul EB. *Resume the electrophoresis for additional 15sec, extract DNA (all the extracted DNA from the same sample are mixed in a tube) *Refill the wells with 20ul EB, and run the electrophoresis for additional 3 minutes. Take a picture of the gel to document the extracted fragment sizes. *Concentrate the DNA with a SpeedVac (no heat) to ~36ul (takes about 1hour). ===Ligation. Blunt-end and TA ligations use the same protocol but slightly different adaptor sequences.=== * Set up ligation reaction. Note that the ATP in the Quick Ligase buffer hydrolyzes very quickly after several rounds of freeze/thaw cycles, so it’s a good idea to make small aliquots of a fresh tube of Quick Ligase Buffer, and use one small aliquot each time. {| {{table}} | ||ul |- | End-repaired & size selected DNA||36 |- | 40uM adaptor2||2 |- | 5X Quick Ligase Buffer||10 |- | Quick Ligase||2 |- | |} *Incubate at room temperature for 15 minutes, purified with MinElute columns, eluted with 20ul EB. ===PCR of sequencing library=== Solexa_PCR_up: AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT AmpR6.3Sol: CAAGCAGAAGACGGCATACGAGCTCTTCGGAACGATGAGCCTCCAAC AmpF6.3rSol: CAAGCAGAAGACGGCATACGAGCTCTTCCAGATGTTATCGAGGTCCGA {| {{table}} | Ligation products||10||10 |- | 10uM solexa PCR up||2||2 |- | 10uM AmpR6.3Sol||2||- |- | 10uM AmpF6.3Sol||-||2 |- | 2X iProof master mix (Bio-Rad)||50||50 |- | 50X SYBR Green I||0.4||0.4 |- | H2O||36||36 |- | |} PCR program: 98 °C 30sec -> 8 cycles of (98°C 10sec -> 60°C 20 sec -> 72°C 15sec) -> 72°C 3min ->15C hold *Mix the amplicons with two sets of primers, purified with Minelute columns. Elute with 31ul EB buffer *Run a TBE gel ===TBE Gel Size Selection=== *Run samples in 3 lanes and ladder in 2 lanes in in a 5-well TBE gel *Load 2 lanes with 25bp ladder Mix in a .2ml tube(30ul H2O+9ul 6X loading dye+1ul 25bp ladder)/2 *Mix (30ul H2O + 15ul 6X loading dye + 15ul sample)/3 load to 3 wells
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