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==Materials== {| {{table}} border=1 | align="center" style="background:#f0f0f0;"|'''Reagents and Kits''' | align="center" style="background:#f0f0f0;"|'''Source''' | align="center" style="background:#f0f0f0;"|'''Cat#''' |- | EZ DNA Methylation Gold Kit||Zymo Research||D5005 |- | 10x Ampligase buffer||Epicentre||A1905B |- | Ampligase||Epicentre||A3210K |- | dNTP||New England Biolab||N0447L |- | Stoffel fragment||Applied Biosystems||N808-0038 |- | ExoI||Epicentre||X40520K |- | ExoIII||Epicentre||EX4425K |- | 2xPhusion HF Master Mix||New England Biolab||F-531L |- | MmeI||New England Biolab||R0637L |- | Quick Ligation Kit||New England Biolab||M2200S |- | SYBR GreenI nucleic acid gel stain||Invitrogen||S7585 |- | Agencourt AMPure XP||Beckman Coulter||A63880 |- | 100% Ethanol || || |- | QIAQuick PCR Purification Kit || Qiagen || 28106 |- | E-Gel Size Select 2% || Invitrogen || G6610-02 |} {| {{table}} border=1 | align="center" style="background:#f0f0f0;"|'''Materials''' | align="center" style="background:#f0f0f0;"|'''Source''' | align="center" style="background:#f0f0f0;"|'''Cat#''' |- | Low Tube Strips, CLR||BioRad||TLS0801 |- | Microseal 'B' Film||BioRad||MSB1001 |- | Barrier Tips, or low retention pipette tips|| Neptune || BT200, BT10E, BT1000, BT20 |- | Mineral Oil|| Sigma|| M5904-500ml |- | 6% TBE Gel 1.0mmx10well ||Invitrogen|| EC6265BOX |- | 10x TBE buffer for PAGE || National Diagnostics || EC-860 |- | SYBR Gold nucleic acid gel stain||Invitrogen|| S11494 |} {| {{table}} border=1 | align="center" style="background:#f0f0f0;"|'''Primers''' | align="center" style="background:#f0f0f0;"|'''Sequence''' | align="center" style="background:#f0f0f0;"|'''Modifications''' |- | AmpF6.3NH2||/5AmMC6/CAGATGTTATCGAGGTCCGAC||5' Amino modifier C6 |- | AmpR6.3NH2||/5AmMC6/GGAACGATGAGCCTCCAAC||5' Amino modifier C6 |- | PE_t_N2, Y-adapter for MmeI ligation-top ||ACACTCTTTCCCTACACGACGCTCTTCCGATCTN*N||3'-Phosphorothioate bond |- | PE_b_A, Y-adapter for MmeI ligation-bottom ||/5Phos/AGATCGGAAGAGCGGTTCAGCAGGAATGCCGAG || 5'-Phosphorylation |- | PCR_Fs|| AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTC || None |- | PCR_R.N2Ind1|| CAAGCAGAAGACGGCATACGAGATCGTGATCTCGGCATTCCTGCTGAACCGCTCTT || None |- | PCR_R.N2Ind2|| CAAGCAGAAGACGGCATACGAGATACATCGCTCGGCATTCCTGCTGAACCGCTCTT || None |- | PCR_R.N2Ind3|| CAAGCAGAAGACGGCATACGAGATGCCTAACTCGGCATTCCTGCTGAACCGCTCTT || None |- | PCR_R.N2Ind4|| CAAGCAGAAGACGGCATACGAGATTGGTCACTCGGCATTCCTGCTGAACCGCTCTT || None |} ==Bisulfite Conversion== Perform bisulfite conversion according to instructions given in EZ Methylation Gold Kit. ==DMR220K Normalization Capture== * gDNA template: bis-cvt BJ-iPS (145.5ng/ul, Nanodrop) ===Calculations=== {| {{table}} border=1 | align="center" style="background:#f0f0f0;"|Probe/Target Ratio||100|| 100 || 100 |- | align="center" style="background:#f0f0f0;"|Probes size||6500|| 27000 || 55000 |- | align="center" style="background:#f0f0f0;"|Template (ng)||200|| 200 || 200 |- | align="center" style="background:#f0f0f0;"|Human gDNA MW (g/mole)||1.82E+012|| 1.82E+012|| 1.82E+012 |- | align="center" style="background:#f0f0f0;"|Human gDNA(moles)||1.10E-019|| 1.10E-019|| 1.10E-019 |- | align="center" style="background:#f0f0f0;"|Probes required (moles)||1.10E-017|| 1.10E-017|| 1.10E-017 |- | align="center" style="background:#f0f0f0;"|Probes MW (g/mole)||2.03E+008|| 8.45E+008 || 1.72E+009 |- | align="center" style="background:#f0f0f0;"|Amount probes (ng)||2.23|| 9.27 || 18.88 |} ===Probes Subset Information=== * Probes were prepared by DD and ZL. * We re-quantified the probes and got higher concentration, the issue was probably not shaking in SYBR Gold solution long enough. ===Experimental Setup=== * We prepared the reactions in PCR tubes * Buffer used is 10x Ampligase Buffer * Set up capture reaction in low profile strip tubes (or PCR tubes/plates). * Spin down the reaction mix and add 1-2 droplets of mineral oil with P200. * Use Microseal film to cover the tubes. * First 7 reactions: Capture with individual probe set. {| {{table}} border=1 | align="center" style="background:#f0f0f0;"|'''Amount Probes''' | align="center" style="background:#f0f0f0;"|'''DMR subset''' | align="center" style="background:#f0f0f0;"|'''Probes(ul)''' | align="center" style="background:#f0f0f0;"|'''Template(ul)''' | align="center" style="background:#f0f0f0;"|'''Buffer(ul)''' | align="center" style="background:#f0f0f0;"|'''H2O(ul)''' | align="center" style="background:#f0f0f0;"|'''Total(ul)''' |- | 2.23ng||DMR.s1||11.08||1.37||2||5.54||20 |- | 2.23ng||DMR.s2||8.81||1.37||2||7.81||20 |- | 18.88||DMR.s3||0.5||1.37||2||16.12||20 |- | 18.88||DMR.s4||3.59||1.37||2||13.04||20 |- | 18.88||DMR.s5||3.05||1.37||2||13.58||20 |- | 9.27||DMR.s6||1.34||1.37||2||15.28||20 |- | 9.27||DMR.s7||2.09||1.37||2||14.53||20 |} '''Program''' 95c 30sec -> cool down to 58C at 0.02C/sec -> 58C 20h -> add 2ul SLN mix(2U/ul AmpliTaq Stoffel fragment; 0.5U/ul AmpLigase; 50uM dNTP) -> 58C 20h-> 94C 2min -> add 2ul Exo I/III mix-> 37C 1h -> 94C 2min -> 4C hold. '''Adding 2ul SLN mix after >20 h of 58C incubation:''' Leave tubes in thermocycler and just remove Microseal film cover to add SLN mix. Use P10, low retention tips and make sure to reach the bottom of the tube before dispensing. Dispense slowly, then slowly pull out the pipette tip before releasing hold on dispenser. Ensure that all reagents were pumped out. Cut new Microseal films to cover the tubes. '''Adding 2ul ExoI/III mix after 3-4 h of 58C incubation:''' Perform same steps as adding SLN. {| {{table}} border="2" | align="center" style="background:#f0f0f0;"|'''SLN Mix''' | align="center" style="background:#f0f0f0;"|''' 1x''' | align="center" style="background:#f0f0f0;"|'''16x''' | align="center" style="background:#f0f0f0;"|'''Final Conc''' |- | H2O||0.55||8.8|| |- | dNTP(1mM)||0.05||0.8||50uM |- | 10xAmpligase Buffer||0.1||1.6||1x |- | AmpLigase||0.1||1.6||0.5U/ul |- | Stoffel||0.2||3.2||2U/ul |- | TOTAL||1||16|| |- |} '''For ExoI/III mix, add equal volumes of Exo I and Exo III.''' ===RT PCR=== {| {{table}} border=1 | align="center" style="background:#f0f0f0;"|'''Reagent''' | align="center" style="background:#f0f0f0;"|'''Concentration''' | align="center" style="background:#f0f0f0;"|'''Vol (ul)''' | align="center" style="background:#f0f0f0;"|'''Final Concentration''' | align="center" style="background:#f0f0f0;"|'''11x Master Mix''' |- | Template||---||10|||| |- | AmpR6.3NH2||10uM||2||0.2uM||22 |- | AmpF6.3NH2||10uM||2||0.2uM|| |- | Phusion HF 2x MM||2x||50||1x||550 |- | SYBR Green||50x||0.4||1x||4.4 |- | H2O||---||36||||396 |- | ||TOTAL||100.4||||972.4 |} '''Program''' 98C 30s -> (98C 10s -> 58C 20s -> 72C 20s)x15 ->72C 3min Purified each reaction with 0.7x AMPure Beads. [[File:ZhangLab 2 2010-10-07 DMR220KNorm.jpg | 300px]] 1 ul of each subset loaded. We combined s1-s7, A, and B to form the barcoded DMR library, then performed PAGE purification. ==Solexa sequencing library construction== # MmeI digestion # Ligation of PE_N2 adapter # PCR with barcoded primers # Combine libraries and perform PAGE purification # Quantification by qPCR
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