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== Experimental procedures == * I firstly dilute CTT gDNA to 2ng/ul volume 50ul :{| {{table}} border = 1 | align="center" style="width:120px;background:#f0f0f0;"|'''Sample''' | align="center" style="width:100px;background:#f0f0f0;"|'''Conc. (ng/ul)''' | align="center" style="width:100px;background:#f0f0f0;"|'''Stock volume (ul)''' | align="center" style="width:100px;background:#f0f0f0;"|'''TE (ul)''' |- | CTT-frozen||52.30||1.91||48.09 |- | CTT-FFPE||64.80||1.54||48.46 |} * For 5ng input, I will add 25pg of unmethylated lambda DNA (add 2.08ul of stock 12pg/ul) * For 100ng input, I will add 0.5ng of unmethylated lambda DNA (add 0.5ul of stock 1ng/ul) === 1) DNA fragmentation with ''Msp''I === ====1.1) Low-input RRBS ''Msp''I digestion set up ==== :{| {{table}} class = wikitable | align="center" style="width:100px;background:#f0f0f0;"|'''CTT-5ng_1''' | align="center" style="width:100px;background:#f0f0f0;"|'''CTT-5ng_2''' | align="center" style="width:100px;background:#f0f0f0;"|'''CTT-F-5ng_1''' | align="center" style="width:100px;background:#f0f0f0;"|'''CTT-F-5ng_2''' | align="center" style="width:100px;background:#f0f0f0;"|'''5ng NTC''' |- |} :{| {{table}} class = wikitable | align="center" style="width:120px;background:#f0f0f0;"|'''Sample''' | align="center" style="width:80px;background:#f0f0f0;"|'''Conc. (ng/ul)''' | align="center" style="width:80px;background:#f0f0f0;"|'''Volume for 5ng (ul)''' | align="center" style="width:80px;background:#f0f0f0;"|'''10X Tango Buffer''' | align="center" style="width:80px;background:#f0f0f0;"|'''MspI (10U/ul)''' | align="center" style="width:80px;background:#f0f0f0;"|'''12pg/ul unmeth-lambda DNA''' | align="center" style="width:80px;background:#f0f0f0;"|'''H2O (ul)''' | align="center" style="width:80px;background:#f0f0f0;"|'''Total (ul)''' |- | 1.1 CTT-frozen||2.00||2.5||2.00||1.00||2.08||10.42||18.00 |- | 1.2 CTT-FFPE||2.00||2.5||2.00||1.00||2.08||10.42||18.00 |- | 1.3 NTC||0.00||2.5||2.00||1.00||2.08||10.42||18.00 |} :<u>'''''Msp''I reaction mix I'''</u> :{| {{table}} border = 1 | align="center" style="width:150px;background:#f0f0f0;"|'''Components''' | align="center" style="width:90px;background:#f0f0f0;"|'''1 rxn''' | align="center" style="width:90px;background:#f0f0f0;"|'''5.5 rxn mix''' |- | MspI (10U/ul)||1.00||5.50 |- | 10X Tango Buffer||2.00||11.00 |- | 12pg/ul unmeth-lambda DNA||2.08||11.44 |- | H2O||10.42||57.31 |- | Total||15.50|| |} :- Aliquot 15.50ul to each tube :- Add 2.5ul of diluted CTT DNA to each tube :- Mix by gentle pulse-vortexting for 10x (put reaction tube on PCR rack) and spin down ====1.2) STD RRBS ''Msp''I digestion set up ==== :{| {{table}} class = wikitable | align="center" style="width:100px;background:#f0f0f0;"|'''CCT-100ng_1''' | align="center" style="width:100px;background:#f0f0f0;"|'''CTT-100ng_2''' | align="center" style="width:100px;background:#f0f0f0;"|'''CTT-F-100ng_1''' | align="center" style="width:100px;background:#f0f0f0;"|'''CTT-F-100ng_2''' | align="center" style="width:100px;background:#f0f0f0;"|'''100ng NTC''' |- |} :{| {{table}} class = wikitable | align="center" style="width:120px;background:#f0f0f0;"|'''Sample''' | align="center" style="width:80px;background:#f0f0f0;"|'''Conc. (ng/ul)''' | align="center" style="width:80px;background:#f0f0f0;"|'''Volume for 100ng (ul)''' | align="center" style="width:80px;background:#f0f0f0;"|'''10X Tango Fuffer''' | align="center" style="width:80px;background:#f0f0f0;"|'''MspI (10U/ul)''' | align="center" style="width:80px;background:#f0f0f0;"|'''H2O to adjust volume (ul)''' | align="center" style="width:80px;background:#f0f0f0;"|'''1ng/ul unmeth-lambda DNA''' | align="center" style="width:80px;background:#f0f0f0;"|'''H2O in MspI mix(ul)''' | align="center" style="width:80px;background:#f0f0f0;"|'''Total (ul)''' |- | 1.1 CTT-frozen||52.30||1.91||2.00||1.00||5.09||0.50||7.50||18.00 |- | 1.2 CTT-FFPE||64.80||1.54||2.00||1.00||5.46||0.50||7.50||18.00 |- | 1.3 NTC||0.00||0.00||2.00||1.00||7.00||0.50||7.50||18.00 |} :<u>'''''Msp''I reaction mix II'''</u> :{| {{table}} border = 1 | align="center" style="width:150px;background:#f0f0f0;"|'''Components''' | align="center" style="width:90px;background:#f0f0f0;"|'''1 rxn''' | align="center" style="width:90px;background:#f0f0f0;"|'''5.5 rxn mix''' |- | MspI (10U/ul)||1.00||5.50 |- | 10X Tango Buffer||2.00||11.00 |- | 1ng/ul unmeth-lambda DNA||0.5||2.75 |- | H2O||7.50||41.25 |- | Total||11.00|| |} :- Add H2O to adjust volume as table above :- Aliquot 11ul to each tube :- Add CTT DNA to each tube following the number in table above :- Mix by gentle pulse-vortexting for 10x (put reaction tube on PCR rack) and spin down --------------------------------------------------------------------------- :- Incubate at 37C for 3hr :- Heat inactivate at 65C for 20min === 3) End-repair/dA-tailing === '''''<span style="color:crimson"><u>Prep</u></span>'''''<br> - To prevent contamination to enzyme tube by multiple time pipetting, I aliquot 11ul each of <u>Klenow fragment, exo-</u> and <u>dA:dC:dG mix</u> into PCR tube.<br> - dA:dC:dG solution was prepared in the concentration (20mM:2mM:2mM). Final concentration in 20ul reaction is 1mM:0.1mM:0.1mM. :- Add 2ul of dA:dC:dG/Klenow fragment exo- to each tube :- Spin down the tube :- Mix by gentle pulse-vortexting for 10x :- Spin down the tube :- Incubate at 30C for 20min (for gap-filling) --> 37C for 20min (for extra dA-tailing) <u>no heat lid will help to protect Klenow fragment, exo</u> :- Heat inactivate enzyme at 75C for 10min :- Set program to hold at 4C :- Spin down the tube before continuing to next step === Methylated adaptor ligation === - To avoid the overlapping of indexes to some scRRBS libraries that may need to sequence in the same run (HiSeq Rapid run has only 2 lanes), I will not use index 2, 4, 5, and 12<br> - I will use 1ul of 1:10 diluted TruSeq adaptors for both low-input RRBS and STD RRBS.<br> '''Index list'''<br> {| {{table}}class = wikitable | align="center" style="width:80px;background:#f0f0f0;"|'''CCT-5ng_1''' | align="center" style="width:80px;background:#f0f0f0;"|'''CTT-5ng_2''' | align="center" style="width:80px;background:#f0f0f0;"|'''CTT-F-5ng_1''' | align="center" style="width:80px;background:#f0f0f0;"|'''CTT-F-5ng_2''' | align="center" style="width:80px;background:#f0f0f0;"|'''5ng NTC''' |- | Ind_9||Ind_10||Ind_11||Ind_13||Ind_14 |} {| {{table}} class = wikitable | align="center" style="width:100px;background:#f0f0f0;"|'''CCT-100ng_1''' | align="center" style="width:100px;background:#f0f0f0;"|'''CTT-100ng_2''' | align="center" style="width:100px;background:#f0f0f0;"|'''CTT-F-100ng_1''' | align="center" style="width:100px;background:#f0f0f0;"|'''CTT-F-100ng_2''' | align="center" style="width:100px;background:#f0f0f0;"|'''100ng NTC''' |- | Ind_15||Ind_16||Ind_18||Ind_19||Ind_20 |} '''''<span style="color:crimson"><u>Prep</u></span>'''''<br> - Diluted 1:10 of TrueSeq methylated adaptors by mixing 1ul of adaptor with 10ul H2O and mix - 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;"|'''11x rxn mix''' |- | dA-tailed reaction||20.00||0.00 |- | 10X Tango buffer||0.50||5.50 |- | HC T4 DNA ligase (30units/ul)||1.00||11.00 |- | 10mM ATP ||1.25||13.75 |- | H2O||1.25||13.75 |- | Total||24.00||44.00 |} :- Add 1ul of diluted methylated adapter :- Add 4ul of ligation reaction mix :- Spin down the tube :- Mix by gentle pulse-vortexting for 10x :- Spin down the tube :- ''Incubate at 16C for 18h (no heat lid)'' :- ''Heat inactivate at 65C for 20min'' '''2014-06-03, continued'''<br> ==== Low-input RRBS ==== :- Heat inactivate at 65C, 20min :- Wait for STD RRBS sample for AMPure bead purification ==== STD RRBS ==== - AMPure bead purification with 2X volume of the bead :- Mix 50ul of AMPure beads with 25ul ligated DNA. Mix by pipetting 10x :- Sit for 30min :- Transfer to sit on magnet for 5min :- Wash twice with 160ul freshly prepared 75% EtOH :- Dry the bead for 3-5min :- Resuspend with 27 H2O :- Transfer to new tube for bisulfite conversion. 25ul will be used for bisulfite conversion. === 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 1 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 10.2 rxn, I mixed 6.12mL of Binding Buffer with 10.2ul of 10ng/ul tRNA (this actually for low-input, but I think it should be fine) : - 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 18min'' :- 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 ---- ----
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