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==Prepare genomic DNA for hybridization== *samples to be prepared: *sample 1: CVF-gDNA *sample 2: CViB-gDNA two sheared gDNA received from Harvard: *sample 3: CD1 PGP1 ips P16 (30ul) *sample 4: PGP1F 8 gDNA (30ul) ===End-repair Reactions=== Fragmented DNA 85 ul 30ul for sample 3,4 10X End Repair Reaction Buffer 10 ul 4ul for sample 3,4 End Repair Enzyme Mix 5 ul 5ul for sample 3,4 Keep the tube at room temperature (~20°C) for 30 minutes. Purify with Qiaquick column and elute in 40ul ddH2O 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. *nanodrop result: *CVf: 45ng/ul *40ul *CD1 PGP1 ips P16: 137.8ng/ul *PGP1F 8 gDNA: 146.3ng/ul * 40ul ===A-Tailing reactions=== Blunt-end DNA 40 ul 10X dA-Tailing Reaction Buffer (10X) 5 ul Klenow Fragment (3’-5’ exo-) 3 ul Incubated at 37C for 30min purified the products with Qiaquick column and elute in 40ul ddH2O ===adaptor ligation=== Prepare adaptors (need to be done only for the first time): 100uM PE-t: 20ul 100uM PE-b: 20ul 10x stoffel buffer: 10ul H2O: 50ul 94C for 3 min, and then cool down to 20C at the rate of 0.1C/sec. commonly used adaptors: Blunt-end adaptors: 5’NH2- ACACTCTTTCCCTACACGACGCTCTTCCGATCT-3’OH Solexa_1_up 3’NH2-ATGTGAGAAAGGGATGTGCTGCGAGAAGGCTAGA-5’OH Solexa_1_lo_nop TA adaptors (for the one adaptor protocol): 5- CAAGCAGAAGACGGCATACGAGCTCTTCCGATCT-3’OH PE_t_adapter 3-CTTCTGCCGTATGCTCGAGAAGGCTAG-5’Phos t_adaptor_rc_s regular Y adaptor: PE_t_adaptor(top) ACACTCTTTCCCTACACGACGCTCTTCCGATC*T 3'-Phosphorothioate bond PE_b_adaptor(bottom) \\5phos\\GATCGGAAGAGCGGTTCAGCAGGAATGCCGAG 5'-phosphorylation 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. adaptor:target molar ratio is 1:15 A-tailed DNA 10 ul for CVF, 3ul for CViB, CD1 PGP1 ips, and PGP1F 8 20uM Y adaptor 3 ul 5X Quick ligase buffer 10 ul Quick Ligase 3 ul H2O 24 ul for CVF, 31ul for CViB, CD1 PGP1 ips, and PGP1F 8 Incubate at room temperature for 15 minutes purify the product with Agencourt AMpure kit and elute in 40ul ddH2O ===PCR=== Ligation products 5ul (2 well for each set, total of 8 well) 100uM Solexa_PCR_up 0.2ul 100uM solexa_PCR-lo 0.2ul 2X phusion HF master mix 50ul 50X SYBR Green I 0.4ul H2O 45ul PCR program: 98 °C 30sec -> 13 cycles of (98°C 10sec -> 60°C 20 sec -> 72°C 15sec) -> 72°C 3min ->15°C hold. purify the products with Qiagen Qiaquick columns and elute in 40ul ddH2O
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