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== Experimental Procedures == ==== Plate layout ==== :{| {{table}} class = wikitable | align="center" style="background:#f0f0f0;" width="50"|'''''' | align="center" style="background:#f0f0f0;" width="50"|'''4''' | align="center" style="background:#f0f0f0;" width="50"|'''5''' | align="center" style="background:#f0f0f0;" width="50"|'''6''' |- | A||1||100||0 |- | B||1||100||0 |- | C||1||20||0 |- | D||1||20||0 |- | E||1||10||0 |- | F||1||10||0 |- | G||1||1||0 |- | H||1||1||0 |} === 1) Cell lysis === '''''<span style="color:crimson"><u>Prep</u></span>'''''<br> - Thaw nuclei from -80C & spin down at 2,000rpm for 5min (96-well plate rotor, 5min)<br> - Mix 30ul of QIAGEN Protease with 1.5ul of 1.2pg/ul lambda DNA :- Add 1.05ul of protease/lambda DNA mix :- Spin down the plate at 2,000rpm for 2min :- Mix by gentle pulse-vortexting for 10x :- Spin down the plate at 2,000rpm for 3min ::- I tried to avoid pipetting up and down to mix the reaction to prevent nuclei/DNA lost. ::- I notice solution was mixed well and no foaming generated ::- I spin down the plate before and after mixing quite long to make sure that all reagents were collected to the bottom of the well :- ''Incubate at 50C for 3hr''<br> :- ''Heat inactivate at 75C for 30min'' :- Set program to hold at 15C :- Spin down the plate at 2,000rpm for 1min before continuing to next step <br> === 2) DNA fragmentation with ''Msp''I === * Incubated released naked DNA with 9units of ''Msp''I in 18ul reaction at 37C for 3hr. '''''<span style="color:crimson"><u>Prep</u></span>'''''<br> - Prepare ''Msp''I reaction mix :{| {{table}} class = wikitable | align="center" style="background:#f0f0f0;"|'''Components''' | align="center" style="background:#f0f0f0;"|'''Volume (ul)''' | align="center" style="background:#f0f0f0;"|'''26x rxn mix''' |- | Lysed nuclei||5.00||0.00 |- | 10X Tango buffer||2.00||52.00 |- | ''Msp''I||0.90||23.40 |- | H2O||10.10||262.60 |- | Total||18.00||338.00 |} - Aliquot 42ul of ''Msp''I enzyme mix to each tube in 8-tube strip :- Add 13ul to each well with multi-channel pipette :- Spin down the plate at 2,000rpm for 2min :- Mix by gentle pulse-vortexting for 10x :- Spin down the plate at 2,000rpm for 3min :- ''Incubate at 37C for 3hr'' :- ''Heat inactivate at 65C for 20min'' '''<NEW>''' :- Set program to hold at 4C :- Spin down the plate at 2,000rpm for 1min before continuing to next step <br> === 3) Gap-filling/dA-tailing === * Add '''5 units of Klenow fragment exo-''', supplemented with '''1mM dATP, 0.1 mM dGTP and 0.1 mM of dCTP''' in 20ul reaction. (Skip dTTP because enzyme cleaves C^CGG) '''''<span style="color:crimson"><u>Prep</u></span>'''''<br> - Prepare new tube of ACG mix {| {{table}} class = wikitable | align="center" style="background:#f0f0f0;"|'''dNTPs''' | align="center" style="background:#f0f0f0;"|'''Stock (mM)''' | align="center" style="background:#f0f0f0;"|'''Dilute to (mM)''' | align="center" style="background:#f0f0f0;"|'''Volume (ul)''' |- | dATP||100||20.00||100.00 |- | dCTP||100||2.00||10.00 |- | dGTP||100||2.00||10.00 |- | H2O (ul)||||||380.00 |- | Total (ul)||||||500.00 |} - Aliquot 5ul of dA:dC:dG mix (20mM:2mM:2mM) to each tube of 8-tube strip<br> - Aliquot 3.5ul of Klenow fragment exo- to each tube of 8-tube strip<br> :- Add 1ul of dA:dC:dG solution mix to each well with multichannel pipette :- Add 1ul of Klenow fragment exo- to each well with multichannel pipette :- Spin down the plate at 2,000rpm for 2min :- Mix by gentle pulse-vortexting for 10x :- Spin down the plate at 2,000rpm for 3min :- ''Incubate at 30C for 20min'' (for gap-filling) --> ''37C for 20min'' (for extra dA-tailing) :- ''Heat inactivate enzyme at 75C for 10min'' :- Set program to hold at 4C :- Spin down the plate at 2,000rpm for 1min before continuing to next step <br> === 4) Methylated adaptor ligation === * Ligate A-tailed DNA with 1ul of 1:20 diluted Illumina indexed methylated adaptor in total reaction 25ul at 16C for 30min and 4C for at least 8h '''Index list'''<br> - I used diluted methylated adaptors prepared on 2014-04-21<br> - Since there was something wrong with Ind_12 in tube_6, I will remove all solution and add 1:20 diluted Ind_5 {| {{table}} class = wikitable | align="center" style="background:#f0f0f0;"|'''Tube_1''' | align="center" style="background:#f0f0f0;"|'''Tube_2''' | align="center" style="background:#f0f0f0;"|'''Tube_3''' | align="center" style="background:#f0f0f0;"|'''Tube_4''' | align="center" style="background:#f0f0f0;"|'''Tube_5''' | align="center" style="background:#f0f0f0;"|'''Tube_6''' | align="center" style="background:#f0f0f0;"|'''Tube_7''' | align="center" style="background:#f0f0f0;"|'''Tube_8''' |- | Ind_2||Ind_4||Ind_5||Ind_6||Ind_7||Ind_5||Ind_2||Ind_4 |} '''''<span style="color:crimson"><u>Prep</u></span>'''''<br> - Mix 1ul of barcoded methylated adaptors Ind_5 with 20ul H2O (on ice box) and add to tube_6<br> - Prepare ligation reaction mix {| {{table}} class =wikitable | align="center" style="background:#f0f0f0;"|'''Components''' | align="center" style="background:#f0f0f0;"|'''Volume (ul)''' | align="center" style="background:#f0f0f0;"|'''28x rxn mix''' |- | dA-tailed reaction||20.00||0.00 |- | 10X Tango buffer||0.50||14.00 |- | HC T4 DNA ligase (30units/ul)||1.00||28.00 |- | 10mM ATP ||1.25||35.00 |- | H2O||1.25||35.00 |- | Total||24.00||112.00 |} - Aliquot 14ul of Ligation mix to each tube in 8-tube strip :- Add 1ul of diluted methylated adapter :- Add 4ul of ligation reaction mix :- Spin down the plate at 2000rpm for 2min :- Mix by gentle pulse-vortexting for 10x :- Spin down the plate at 2,000rpm for 3min :- ''Incubate at 16C for 30min -> 4C for 12h (no heat lid)'' :- ''Heat inactivate at 65C for 20min'' :- Spin down the plate at 2,000rpm for 1min before continuing to next step :{| {{table}} class = wikitable | align="center" style="background:dodgerblue;"|'''Well ID''' | align="center" style="background:magenta;"|'''Index''' | align="center" style="background:springgreen;"|'''Sample #''' | align="center" style="background:dodgerblue;"|'''Well ID''' | align="center" style="background:magenta;"|'''Index''' | align="center" style="background:springgreen;"|'''Sample #''' | align="center" style="background:dodgerblue;"|'''Well ID''' | align="center" style="background:magenta;"|'''Index''' | align="center" style="background:springgreen;"|'''Sample #''' |- | A4||Ind_2||#1||A5||Ind_2||#9||A6||Ind_2||#17 |- | B4||Ind_4||#2||B5||Ind_4||#10||B6||Ind_4||#18 |- | C4||Ind_5||#3||C5||Ind_5||#11||C6||Ind_5||#19 |- | D4||Ind_6||#4||D5||Ind_6||#12||D6||Ind_6||#20 |- | E4||Ind_7||#5||E5||Ind_7||#13||E6||Ind_7||#21 |- | F4||Ind_5||#6||F5||Ind_5||#14||F6||Ind_5||#22 |- | G4||Ind_2||#7||G5||Ind_2||#15||G6||Ind_2||#23 |- | H4||Ind_4||#8||H5||Ind_4||#16||H6||Ind_4||#24 |} '''2014-04-24''' === 5) Bisulfite conversion === * I performed bisulfite conversion using the same procedure following manufacturer's instruction and elute with 31ul elution buffer. '''''<span style="color:crimson"><u>Prep</u></span>'''''<br> - Prepare 3 tubes of CT Conversion Reagent, by adding 850ul H2O, 50ul of Resuspension Buffer, and 300ul of Dilution Buffer to CT Conversion Reagent (for 25ul DNA sample --> reduce H2O from 900 to 850) :- Add125ul of complete CT Conversion Reagent to adaptor ligated DNA (no sample transfer to the new tube) :- Mix by pipetting 10X with multi-channel pipette :- Spin down the plate at 2,000rpm for 1min :- Incubate following below program ::- 98°C for 10 minutes (DNA denaturation) ::- 64°C for 2.5 hours (Bisulfite conversion) ::- 4°C storage for up to 20 hours or continue to desulfonation '''''<span style="color:crimson"><u>Prep</u></span>'''''<br> - Mix 600:1 ratio of Binding Buffer and 10ng/ul tRNA<br> - For 24 rxn, I mixed 14.7mL of Binding Buffer with 24.5 ul of 10ng/ul tRNA : - Add 601ul of Binding Buffer/tRNA mix to the column :- Bind DNA to column by transfer bisulfite-treated DNA to the column and mixing by pipetting up and down for 5X. I rinse the well with small amount of Binding Buffer to transfer DNA to the column as much as possible :- Spin down 14,000rpm for 30sec. <u>Discard spnt</u> :- Wash with 100ul Wash buffer :- Spin down 14,000rpm for 30sec :- ''Incubate with 200ul of Desulphonation Buffer for 15min'' :- Spin down 14,000rpm for 30sec :- Wash column with 200ul Wash Buffer. :- Spin down 14,000rpm for 30sec. <u>Discard spnt</u> :- Wash the column with 200ul Wash Buffer. :- Spin down 14,000rpm for 2min :- Elute converted DNA with warm (~60C) 31ul Elution Buffer. Incubate column with Elution Buffer at room temp for 2min. :- Spin down at 14,000rmp for 1min. This should have ~30ul DNA left for PCR === 6) PCR amplification === '''''<span style="color:crimson"><u>Prep</u></span>'''''<br> - Prepare 10uM of TruS_F and TruS_R mix volume 500ul by mixing 50ul each of 100uM TruS_F and 100uM TruS_R with 400ul H2O ==== <u>1st round PCR</u> ==== '''''<span style="color:crimson"><u>Prep</u></span>'''''<br> :{| {{table}} class = wikitable | align="center" style="background:#f0f0f0;"|'''Components''' | align="center" style="background:#f0f0f0;"|'''Conc''' | align="center" style="background:#f0f0f0;"|'''unit''' | align="center" style="background:#f0f0f0;"|'''Final conc.''' | align="center" style="background:#f0f0f0;"|'''unit''' | align="center" style="background:#f0f0f0;"|'''Volume (ul)''' | align="center" style="background:#f0f0f0;"|'''26 rxn mix''' |- | Bis-cvt DNA||||||||||30.00||0.00 |- | 10X Reaction buffer||10||X||1||X||5.00||130.00 |- | dNTP mix||10||mM||0.2||mM||1.00||26.00 |- | TruS_F/R||10||uM||0.3||uM||1.50||39.00 |- | PfuTurbo Cx||2.5||Unit/ul||1||unit||0.40||10.40 |- | 50X SYBG||50||X||0.8||X||0.80||20.80 |- | H2O||||||||||11.30||293.80 |} :- Aliquot 20ul, add 30ul of bisulfite-treat adaptor-ligated DNA ::''95C for 2min --> [95C for 20sec -> '''60C for 30sec''' -> 72C for 1min] X 25 cycles --> 72C for 2min''<br> :- Purified the 1st round amplicons with AMPure beads 2X (1:1 ratio) ==== <u>AMPure bead purification</u> ==== '''''<span style="color:crimson"><u>Prep</u></span>'''''<br> - Freshly prepare 20mL of 75% EtOH by mixing 15mL of 100% EtOH with 5mL of H2O<br> - Add ~2.5mL of resuspened AMPure bead in 30mL reservoir :- Add 50ul AMPure bead. Mix by pipetting 10x :- Sit for 8min :- Transfer to sit on magnet for 5min :- Wash twice with 160ul freshly prepared 80% EtOH :- Dry the bead for 3-5min :- Resuspend with 50 H2O :- Add 50ul of fresh AMPure bead. Mix by pipetting 10x :- Sit for 5min :- Transfer to sit on magnet for 5min :- Wash twice with 160ul freshly prepared 80% EtOH :- Dry the bead for 3-5min <u>(make sure that the beads are completely dried out to avoid EtOH inhibiting PCR)</u> :- Resuspend the bead with 40ul H2O :- Transfer purified 1st round amplicons to 8-tube strip with cap :- Sit the strip tube on magnet before adding to the 2nd round PCR to avoid bead contamination in PCR ::- 32ul of bead purified 1st round amplicons will be added to the 2nd round PCR (32/40 -> 40%) ::- I saved the rest of bead purified 1st round amplicons ==== <u>2nd round PCR</u> ==== :{| {{table}} class = wikitable | align="center" style="background:#f0f0f0;"|'''Components''' | align="center" style="background:#f0f0f0;"|'''Conc''' | align="center" style="background:#f0f0f0;"|'''unit''' | align="center" style="background:#f0f0f0;"|'''Final conc.''' | align="center" style="background:#f0f0f0;"|'''unit''' | align="center" style="background:#f0f0f0;"|'''Volume (ul)''' | align="center" style="background:#f0f0f0;"|'''26 rxn mix''' |- | Purified 1st round DNA||||||||||32.00||0.00 |- | 5X Phusion HF buffer||5||X||1||X||10.00||260.00 |- | dNTP mix||10||mM||0.2||mM||1.00||26.00 |- | TruS_F/R||10||uM||0.3||uM||1.50||39.00 |- | 50X SYBR||50||X||0.8||X||0.80||20.80 |- | Phusion HF||2||unit/ul||||||0.50||13.00 |- | H2O||||||||||4.20||109.20 |} :- Aliquot 18ul, add 32ul of bead purified 1st round amplicons<br> ::''98C for 2min --> [98C for 10sec -> '''60C for 30sec''' -> 72C for 1min] X 22 cycles --> 72C for 2min''<br> :- I plan to monitor 100nu and 20nu to stop them earlier since the amplification should be too saturated at 22cycles of the 2nd round PCR.
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