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= RRBS library preparation of colon tumor tissue samples from Biochain (STD RRBS, 100ng input DNA) = [[noi:DMR220k_LabNotes|'''Calendar''']] * After I have tried STD protocol to get it worked in 12878 sample, I continued to work on CTT samples. * The main things to concern are concentration of adaptor concentration in ligation reaction and optimization of PCR cycle number to prevent over-amplification of sequencing libraries. * In this experiment, I spiked in 10pg of unmethylated lambda DNA which accounts for 0.01% of input DNA. This might be too low. == Experimental procedures == * Sample list ** CTT-frozen, 100ng, two replicates ** CTT-FFPE, 100ng, two replicates === 1) MspI digestion === * Conditions: Digest 100ng of DNA with 20U of MspI in 30ul reaction at 37C 3h and heat inactivate at 65C 10min {| {{table}} class = wikitable | align="center" style="width:140px;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;"|'''12pg/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||3.00||2.00||12.26||0.83||10.00||30.00 |- | 1.2 CTT-FFPE||64.80||1.54||3.00||2.00||12.63||0.83||10.00||30.00 |- | 1.3 NTC||0.00||0.00||3.00||2.00||14.17||0.83||10.00||30.00 |} ==== <u>Msp I reaction Mix</u> ==== {| {{table}} border = 1 | align="center" style="background:#f0f0f0;"|'''Components''' | align="center" style="width:80px;background:#f0f0f0;"|'''1 rxn''' | align="center" style="width:80px;background:#f0f0f0;"|'''5.5 rxn mix''' |- | MspI (10U/ul)||2.00||11.00 |- | 10X Tango Buffer||3.00||16.50 |- | 12pg/ul unmeth-lambda DNA||0.83||4.57 |- | H2O||10.00||55.00 |- | Total||15.83|| |} :- Add H2O to each tube :- Add 100ng of DNA to each tube :- Add 15.83ul of MspI reaction Mix to each tube and pipette few times :- Spin down the tubes :- Mix by gentle pulse-vortexing on PCR rack 10x :- Spin down the tubes :- 37C, 12h --> 65C, 20min --> hold at 4C (set heat lid) :- Note: For this experiment, I set up ''Msp''I at night, so I incubate O/N. Longer incubation did not increase digestion that much. However, it has been reported that it does not cause star activity. === 2) End-repair/dA-tailing === '''''<span style="color:crimson"><u>Prep</u></span>'''''<br> - Mix 6.5ul of Klenow exo- with 6.5ul of dA:dC:dG (stock 20mM:2mM:2mM) just right before adding to ''Msp''I-digested DNA :- Add 2ul of Klwnow, exo- and dA:dC:dG mix to each tube of MspI digested DNA. Pipette few times :- Spin down the tube :- Mix by gentle pulse-vortexing on PCR rack 10x :- Spin down the tube :- 30C, 20min --> 37C, 20min --> hold at 4C (no heat lid) :- 75C, 10min --> hold at 4C (heat lid on) * Conditions: Add 1ul of non-diluted TruSeq adaptors (do not know actual concentration of adaptors) in 30ul ligation reaction with 6000U of T4 DNA ligase * Before set up ligation reaction, purify end-repaired DNA with 2x volume AMPure beads (64ul each) and resuspend the bead with 20ul EB buffer ==== <u>AMPure bead purification</u> ==== '''''<span style="color:crimson"><u>Prep</u></span>'''''<br> - Freshly prepare 5mL of 75% EtOH by mixing 3.75mL of 100% EtOH with 1.25mL of H2O (prepare when the tubes sit on magnet)<br> - Aliquot 710ul of AMPure beads in 1.5mL tube and leave at RT<br> :- Add 64ul AMPure bead. Mix by pipetting 10x :- Wait for 30min :- Transfer to sit on magnet for 5min :- Wash twice with 180ul freshly prepared 75% EtOH ::- After adding 75% EtOH (1st time), wait for 30sec before pipetting up & down 5x :- Remove all 75% EtOH :- Dry the bead for 20min :- Resuspend with 20 EB Buffer <u>''(keep the beads in the tubes!)''</u> === 3) Methylated adaptor ligation === * Conditions: Add 1ul of non-diluted TruSeq adaptors (do not know actual concentration of adaptors) in 30ul ligation reaction with 6000U of T4 DNA ligase * Before set up ligation reaction, purify end-repaired DNA with 2x volume AMPure beads (64ul each) and resuspend the bead with 20ul EB buffer ==== <u>AMPure bead purification</u> ==== '''''<span style="color:crimson"><u>Prep</u></span>'''''<br> - Freshly prepare 5mL of 75% EtOH by mixing 3.75mL of 100% EtOH with 1.25mL of H2O (prepare when the tubes sit on magnet)<br> - Aliquot 325ul of AMPure beads in 1.5mL tube and leave at RT<br> :- Add 64ul AMPure bead. Mix by pipetting 10x :- Wait for 30min :- Transfer to sit on magnet for 5min :- Wash twice with 180ul freshly prepared 75% EtOH ::- After adding 75% EtOH (1st time), wait for 30sec before pipetting up & down 5x :- Remove all 75% EtOH :- Dry the bead for 20min :- Resuspend with 20 EB Buffer <u>''(keep the beads in the tubes!)''</u> ====<u> Ligation reaction Mix</u> ==== {| {{table}} border = 1 | align="center" style="background:#f0f0f0;"|'''Compinents''' | align="center" style="width:80px;background:#f0f0f0;"|'''1x rxn''' | align="center" style="width:80px;background:#f0f0f0;"|'''5.2x rxn''' |- | dA-tailed DNA||20.00||0.00 |- | 10X Ligation Buffer||3.00||15.60 |- | T4 DNA ligase||1.00||5.20 |- | H2O||5.00||26.00 |- | total||29.00|| |} :- Add 1ul of non-diluted TruSeq adaptors and pipette few times to make sure adaptors were added to the reaction :- Add 9ul of ligation reaction mix :- Spin down the tube :- Mix by gentle pulse-vortexing on PCR rack 10x :- Spin down the tube :- 16C, 20h (no heat lid) :- Mix by gentle pulse-vortexing on PCR rack 5x :- Quickly spin down :- Heat inactivate at 65C, 20min --> hold at 4C (heat lid on) * Before continue to bisulfite conversion, purify adaptor-ligated DNA with 2x volume AMPure beads (60ul each) and resuspend the bead with 31ul EB buffer {| {{table}} class = wikitable | align="center" style="width:140px;background:#f0f0f0;"|'''Sample''' | align="center" style="width:80px;background:#f0f0f0;"|'''TruSeq Index''' |- | CTT-frozen-100ng_1||Ind_18 |- | CTT-frozen-100ng_2||Ind_19 |- | CTT-FFPE-100ng_1||Ind_20 |- | CTT-FFPE-100ng_2||Ind_21 |- | NTC||Ind_22 |} ==== <u>AMPure bead purification</u> ==== '''''<span style="color:crimson"><u>Prep</u></span>'''''<br> - Freshly prepare 4mL of 75% EtOH by mixing 3mL of 100% EtOH with 1mL of H2O (prepare when the tubes sit on magnet)<br> - Aliquot 155ul of AMPure beads and 155ul of 20% PEG 8000/5M NaCl in 1.5mL tube, mix well and leave at RT<br> :- Add 60ul AMPure bead. Mix by pipetting 10x :- Wait for 30min :- Transfer to sit on magnet for 5min :- Wash twice with 180ul freshly prepared 75% EtOH ::- After adding 75% EtOH (1st time), wait for 30sec before pipetting up & down 5x :- Remove all 75% EtOH :- Dry the bead for 20min :- Resuspend with 31 EB Buffer and transfer 30ul of adaptor-ligated DNA to new strip tubes <u>(discard the beads)</u> :- Continue to bisulfite conversion === 4) Bisulfite conversion === * Performed bisulfite conversion using the same procedure following manufacturer's instruction [[http://tools.lifetechnologies.com/content/sfs/manuals/methylcode_bisulfite_man.pdf]] and elute with 38ul Elution buffer. '''''<span style="color:crimson"><u>Prep</u></span>'''''<br> :- Mix well and spin down the tube :- Put on thermomixer at 37C for 15min, 1000rpm :- Solution should be clear :- Spin down the tube :- Note: I used CT conversion resuspended on 2014-06-11 ------------------ :- Add 120ul complete CT Converson Reagent to 30ul bead-purified adaptor ligated DNA --> total volume 150ul :- Mix by pipetting 10X with multi-channel pipette :- Spin down the tube :- 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 :- Add 600ul Binding Buffer to each column :- Adjust p200 to 200ul and transfer bisulfite treated DNA to the column :- Mix by pipetting 5x :- Spin down column at 14,000 rpm for 30s :- <u>Discard spnt</u> :- Add 100ul Wash Buffer (Aliquot Wash Buffer in 5mL tube) :- Spin down column at 14,000 rpm for 30s <u>(do not discard spnt)</u> :- Add 200ul Desulfonation Buffer (aliquot Desulfonation Buffer in 2mL or 5mL tube) :- Spin down column at 14,000 rpm for 30s <u>(do not discard spnt)</u> :- Add 200ul Wash Buffer :- Spin down column at 14,000 rpm for 30s :- <u>Discard spnt</u> :- Add 200ul Wash Buffer :- Spin down column at 14,000 rpm for 3min <u>(do not discard spnt)</u> :- Transfer column to prepared 1.5mL LoBind tube & make sure that column does not touch spnt in the collecting tube before transfering to new tube :- Add 38ul of Elution Buffer (warm Elution Buffer at 60C for 10min) :- Wait for 2min :- Spin down column at 12,000 rpm for 1min :- Continue to amplification (During elution step, PCR mix should be prepared) == Amplification == === TEST PCR === {| {{table}} border = 1 | align="center" style="width:140px;background:#f0f0f0;"|'''Components''' | align="center" style="width:80px;background:#f0f0f0;"|'''Conc''' | align="center" style="width:60px;background:#f0f0f0;"|'''unit''' | align="center" style="width:80px;background:#f0f0f0;"|'''Final conc.''' | align="center" style="width:60px;background:#f0f0f0;"|'''unit''' | align="center" style="width:80px;background:#f0f0f0;"|'''Volume (ul)''' | align="center" style="width:80px;background:#f0f0f0;"|'''6.5 rxn mix''' |- | Bis-cvt DNA||||||||||6.00||0.00 |- | 10X Reaction buffer||10||X||1||X||2.00||13.00 |- | dNTP mix||10||mM||0.25||mM||0.50||3.25 |- | TruS_F/R||10||uM||0.3||uM||0.60||3.90 |- | PfuTurbo Cx||2.5||Unit/ul||2.5||unit||0.40||2.60 |- | 50X SYBG||50||X||0.8||X||0.32||2.08 |- | H2O||||||||||10.18||66.17 |} :- Aliquot 14 + 6ul DNA template :- Mix well. Split 2X of 9ul :: ''95C for 2min --> [95C for 20sec -> 60C for 30sec -> 72C for 1min] X 18 cycles --> 72C for 2min'' :- Stopped at 15X and 17X :- Added 10ul 2X loading dye to each strip tube & mix well :- Load 10ul PCR/dye mix in 12% gel and run at 250 volts for 40min (running in regular 6% TBE gel at 250 volts, 25 min is okay) [[File:ZhangLab_2 2014-06-16 13hr 10min_CTT-RRBS-test15x-17X.jpg| 500px]] :- Determine # of cycles based on the intensity of the gel. For CTT-frozen samples, I would stop at 15X . For CTT-FFPE samples which have a faint smear, I would stopped at 17X. * Then set up reaction 100ul for each samples with 30ul BIS-DNA and split into 2X of 50ul to avoid bias from amplification (Ref. split 4X of 25ul) {| {{table}} border = 1 | align="center" style="width:140px;background:#f0f0f0;"|'''Components''' | align="center" style="width:80px;background:#f0f0f0;"|'''Conc''' | align="center" style="width:60px;background:#f0f0f0;"|'''unit''' | align="center" style="width:80px;background:#f0f0f0;"|'''Final conc.''' | align="center" style="width:60px;background:#f0f0f0;"|'''unit''' | align="center" style="width:80px;background:#f0f0f0;"|'''Volume (ul)''' | align="center" style="width:80px;background:#f0f0f0;"|'''8.5 rxn mix''' |- | Bis-cvt DNA||||||||||15.00||0.00 |- | 10X Reaction buffer||10||X||1||X||5.00||42.50 |- | dNTP mix||10||mM||0.25||mM||1.25||10.63 |- | TruS_F/R||10||uM||0.3||uM||1.50||12.75 |- | PfuTurbo Cx||2.5||Unit/ul||2.5||unit||1.00||8.50 |- | 50X SYBG||50||X||0.8||X||0.80||6.80 |- | H2O||||||||||25.45||216.33 |} :- I did not include NTC as it clear in TEST PCR :- Aliquot 70 + 30ul DNA template :- Mix well. Split 2X of 50ul (actual ~48ul) :: ''95C for 2min --> [95C for 20sec -> 60C for 30sec -> 72C for 1min] X 18 cycles --> 72C for 2min'' :- Stopped at 15X for CTT-frozen and 17X for CTT-FFPE :- I took 5ul of PCR product from each tube and mix with 5ul of 2X loading buffer to verify that PCR work (even PCR curve looked fine) :- I pooled the two PCR replicatea (total ~87ul for each samples) and purified with 1x volume AMPure beads and eluted with 50ul EB buffer :- I did PAGE quantification by loading 4ul of bead-purified sequencing libraries [[File:ZhangLab_2 2014-06-16 17hr 52min_CTT-RRBS_100ng_PQ.jpg| 500px]] ==== PAGE quantification results ==== {| {{table}} class = wikitable | align="center" style="background:#f0f0f0;"|'''Sample''' | align="center" style="width:80px;background:#f0f0f0;"|'''TruSeq Index''' | align="center" style="width:80px;background:#f0f0f0;"|'''Conc. (ng/ul)''' | align="center" style="width:80px;background:#f0f0f0;"|'''Amount in 45ul (ng)''' | align="center" style="width:80px;background:#f0f0f0;"|'''Volume for 35ng (ul)''' |- | CTT-frozen-100ng_1||Ind_18||4.80||215.85||7.30 |- | CTT-frozen-100ng_2||Ind_19||4.64||208.89||7.54 |- | CTT-FFPE-100ng_1||Ind_20||1.05||47.31||33.29 |- | CTT-FFPE-100ng_2||Ind_21||1.77||79.63||19.78 |} :- I pooled 35ng each library based on PAGE quantification and did size selection in 2 well of 5-well 6% TBE gel [[File:ZhangLab_2 2014-06-17 14hr 06min_100ng_CTTRRBS_PAGE-SS.jpg| 350px]] :- I precipitated in 3tubes at -80C O/N and resuspended each tube with 11ul H2O --> total volume 33ul :- I took 4ul for PAGE verification. ==== PAGE verification ==== [[File:ZhangLab_2 2014-06-24 17hr 06min_RRBS_PAGE-verify.jpg| 500px]] '''Sequencing library ID: NP-RRBS-CTT-100ng, Jun13, 2014'''<br> - Length: 160-360bp<br> - Conc. by Qubit: XX ng/ul or XX nM
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