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= Preparation of RRBS sequencing libraries of plasma DNA from colon and lung cancer patients = [[noi:DMR220k_LabNotes| '''Calendar''']] ==== Sample info ==== * [[noi:Samples_from_Moores_Cancer_Center#2014-06-26| Plasma from colon cancer patients (006-P)]] * [[noi:Samples_from_Moores_Cancer_Center#2014-06-26| Plasma from lung cancer patients (007-P)]] {| {{table}} class = wikitable | align="center" style="width:120px;background:#f0f0f0;"|'''Project ID''' | align="center" style="width:80px;background:#f0f0f0;"|'''Zhang lab ID''' | align="center" style="width:80px;background:#f0f0f0;"|'''Conc. (ng/ul)''' |- | UCSD-006.1||6-P_1||0.200 |- | UCSD-006.2||6-P_2||1.100 |- | UCSD-006.3||6-P_3||1.570 |- | UCSD-006.4||6-P_4||0.889 |- | UCSD-006.5||6-P_5||0.487 |- | UCSD-006.6||6-P_6||0.249 |- | UCSD-006.7||6-P_7||0.247 |- | UCSD-006.8||6-P_8||0.226 |- | UCSD-006.9||6-P_9||0.408 |- | UCSD-006.10||6-P_10||0.333 |} {| {{table}} class = wikitable | align="center" style="width:120px;background:#f0f0f0;"|'''Project ID''' | align="center" style="width:80px;background:#f0f0f0;"|'''Zhang lab ID''' | align="center" style="width:80px;background:#f0f0f0;"|'''Conc. (ng/ul)''' |- | UCSD-007.1||7-P_1||0.692 |- | UCSD-007.2||7-P_2||1.140 |- | UCSD-007.3||7-P_3||0.208 |- | UCSD-007.4||7-P_4||0.202 |- | UCSD-007.5||7-P_5||1.110 |- | UCSD-007.6||7-P_6||0.233 |- | UCSD-007.7||7-P_7||0.229 |- | UCSD-007.8||7-P_8||0.167 |- | UCSD-007.9||7-P_9||0.231 |- | UCSD-007.10||7-P_10||0.203 |} * X-Y_Z: ** X = project ID number. 4 = pancreas, 6 = colon, 7 = lung ** Y = sample type. T = tissue, P = plasma, S = serum ** Z = sample ID == 1) MspI digestion == * Note that the protocol of low-input DNA (1-5ng) will be different from the STD RRBS protocol (100ng). * Conditions: Digest 1ng of DNA with 10U of MspI in 18ul reaction at 37C O/N (normally 3h) and heat inactivate at 65C 20min * For 1ng DNA input, I used 1ul (10U) of MspI. * I spike in 1% of unmeth lambda DNA (10pg) * I actually start MspI at late night of 2014-06-29, so I incubated it O/N as it doesn't hurt to incubate DNA with MspI longer than 3h.<br> <br> ==== <u>Msp I Reaction Mix</u> ==== {| {{table}} border = 1 | align="center" style="background:#f0f0f0;"|'''Components''' | align="center" style="width:80px;background:#f0f0f0;"|'''1x rxn''' | align="center" style="width:80px;background:#f0f0f0;"|'''26 rxn Mix''' |- | MspI (10U/ul)||1.00||26.00 |- | 10x Tango Buffer||2.00||52.00 |- | 12pg/ul unmeth-lambda DNA||0.83||21.67 |- | H2O||8.17||212.33 |- | Total||12.00|| |} '''''<span style="color:crimson"><u>Prep</u></span>'''''<br> - Aliquot 39ul of MspI reaction mix to 8-tube strip. :- Add H2O to each tube :- Add 1ng of DNA to each tube :- Add 12ul 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, O/N (~12.5h) --> 65C, 20min --> hold at 4C (set heat lid)<br> <br> == 2) End-repair/dA-tailing == '''''<span style="color:crimson"><u>Prep</u></span>'''''<br> - Mix 29ul of Klenow exo- with 29ul of dA:dC:dG (stock 20mM:2mM:2mM) just right before adding to MspI digested DNA<br> - Aliquot 7ul of Klenow, exo- and dA:dC:dG mix to 8-tube strip. :- Add 2ul of Klenow, exo- and dA:dC:dG mix to each tube of MspI digested DNA with multichannel pipette. 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) <br> == 3) Methylated adaptor ligation == * Conditions: Add 1ul of 1:20 diluted TruSeq adaptors (do not know actual concentration of adaptors) in 25ul ligation reaction with 6000U of T4 DNA ligase ==== <u>Ligation reaction set up</u> ==== '''''<span style="color:crimson"><u>Prep</u></span>'''''<br> - Thaw diluted TruSeq adaptor on ice (used the diluted adaptors prepared on 2014-06-20, keep it on ice all the time)<br> - Prepare ligation reaction mix ====<u> Ligation reaction Mix</u> ==== :{| {{table}} border = 1 | align="center" style="width:140px;background:#f0f0f0;"|'''Components''' | align="center" style="width:70px;background:#f0f0f0;"|'''1x rxn''' | align="center" style="width:70px;background:#f0f0f0;"|'''26x rxn''' |- | dA-tailed DNA||20.00||0.00 |- | 10X Tango buffer||0.50||13.00 |- | HC T4 DNA ligase (30units/ul)||1.00||26.00 |- | 10mM ATP ||1.25||32.50 |- | H2O||1.25||32.50 |- | Total||24.00|| |} '''''<span style="color:crimson"><u>Prep</u></span>'''''<br> - Aliquot 13ul of ligation reaction mix to 8-tube strip. :- Add 1ul of 1:20 diluted TruSeq adaptors and pipette few times to make sure adaptors were added to the reaction :{| {{table}} class = wikitable | align="center" style="width:90px;background:dodgerblue;"|'''Sample''' | align="center" style="width:70px;background:magenta;"|'''TruSeq Index''' | align="center" style="width:90px;background:dodgerblue;"|'''Sample''' | align="center" style="width:70px;background:magenta;"|'''TruSeq Index''' |- | 6-P_1||13||7-P_1||1 |- | 6-P_2||14||7-P_2||2 |- | 6-P_3||15||7-P_3||4 |- | 6-P_4||16||7-P_4||5 |- | 6-P_5||27||7-P_5||6 |- | 6-P_6||18||7-P_6||7 |- | 6-P_7||19||7-P_7||8 |- | 6-P_8||20||7-P_8||10 |- | 6-P_9||21||7-P_9||11 |- | 6-P_10||22||7-P_10||12 |- | 6-P_NTC1||22||7-P_NTC1||12 |- | 6-P_NTC2||22||7-P_NTC2||12 |} :- Add 4ul of ligation reaction mix with multichannel pipette :- Spin down the tube :- Mix by gentle pulse-vortexing on PCR rack 10x :- Spin down the tube :- 16C, 22h (no heat lid. Ligation time is flexible. I usually do at least 20h at 16C) :- Mix by gentle pulse-vortexing on PCR rack 5x :- Quickly spin down :- Heat inactivate at 65C, 20min --> hold at 4C (heat lid on)<br> <br> == Bisulfite conversion == * Performed bisulfite conversion using LifeTech MethylCode™ Bisulfite Conversion Kit and did the same procedure following manufacturer's instruction and elute with 38ul Elution buffer. '''''<span style="color:crimson"><u>Prep</u></span>'''''<br> :- Prepare 3 tubes of complete CT Conversion Reagent by adding 850ul H2O, 50ul Resuspension Buffer, and 300ul Dilution Buffer. If CT Conversion reagent prepare with 800ul H2O, need to add 5ul of H2O to 25ul adaptor-ligated DNA and added 120ul of CT conversion reagent. :- Mix well and spin down the tube :- Put on thermomixer at 37C for 15min, 1,000rpm :- Solution should be clear :- Spin down the tube :- Add 125ul 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 transferring to new tube :- Add 30ul 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)<br> <br> == Amplification == === 1st round PCR (fix 12 cycles) === {| {{table}} border = 1 | align="center" style="width:140px;background:#f0f0f0;"|'''Components''' | align="center" style="width:80px;background:#f0f0f0;"|'''Conc''' | align="center" style="width:60px;px;background:#f0f0f0;"|'''unit''' | align="center" style="width:80px;background:#f0f0f0;"|'''Final conc./amount''' | align="center" style="width:60px;px;background:#f0f0f0;"|'''unit''' | align="center" style="width:80px;background:#f0f0f0;"|'''Volume (ul)''' | align="center" style="width:80px;background:#f0f0f0;"|'''25 rxn mix''' |- | Bis-cvt DNA||||||||||30.00||0.00 |- | 10X Reaction buffer||10||X||1||X||5.00||125.00 |- | dNTP mix||10||mM||0.25||mM||1.25||31.25 |- | TruS_F/R||10||uM||0.3||uM||1.50||37.50 |- | 50X SYBG||50||X||0.8||X||0.80||20.00 |- | PfuTurbo Cx||2.5||Unit/ul||1||unit||1.00||25.00 |- | H2O||||||||||10.45||261.25 |- | Total ||||||||||50.00|| |} :- Aliquot 20 + 30ul BIS-DNA template :- Mix well :: ''95C for 2min --> [95C for 20sec -> 60C for 30sec -> 72C for 1min] X 12 cycles --> 72C for 2min'' :- Purify with 1X AMPure beads (incubate DNA with beads 15min, the rest steps are the same as previous experiment) :- Elute with 40ul EB Buffer :- Continue to 2nd round PCR. No PAGE verification. === 2nd round PCR === '''OPTIMIZE PCR cycle number''' * I only TEST 9 cycles of 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;"|'''26 rxn mix''' |- | Purified 1st round DNA||||||||||3.00||0.00 |- | 5X Phusion HF buffer||5||X||1||X||2.00||52.00 |- | dNTP mix||10||mM||0.25||mM||0.25||6.50 |- | TruS_F/R||10||uM||0.3||uM||0.30||7.80 |- | 50X SYBR||50||X||0.4||X||0.08||2.08 |- | Phusion HF||2||unit/ul||||||0.10||2.60 |- | H2O||||||||||4.27||111.02 |- | Total||||||||||10.00|| |} :- Aliquot 7 + 3ul DNA template :- Mix well. ::''98C for 2min --> [98C for 10sec -> '''60C for 30sec''' -> 72C for 1min] X 9X cycles --> 72C for 2min''<br> :- 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 [[File:ZhangLab_2 2014-07-02 14hr 25min_1ngRRBS_6P_1-10_TEST-9x.jpg| 500px]] [[File:ZhangLab_2 2014-07-02 14hr 25min_1ngRRBS_7P_1-10_TEST-9x.jpg| 500px]] * A smear for sample 6P_1-10 did not look very good for me since it 's supposed to be very sharp compare to previous experiment. I think it might be over-amplified. That's why there are s smear above the expected ranged. * I think 8 cycles should reduce background and increase the intensity of a smear between 150-350bp. However, I'm afraid to amplified in all of template DNA at once. I do amplification 1Xrxn of 50ul instead of 2X of 50ul as usual. {| {{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;"|'''20.5 rxn mix''' |- | Purified 1st round DNA||||||||||15.00||0.00 |- | 5X Phusion HF buffer||5||X||1||X||10.00||205.00 |- | dNTP mix||10||mM||0.25||mM||1.25||25.63 |- | TruS_F/R||10||uM||0.3||uM||1.50||30.75 |- | 50X SYBR||50||X||0.4||X||0.40||8.20 |- | Phusion HF||2||unit/ul||||||0.50||10.25 |- | H2O||||||||||21.35||437.68 |- | Total||||||||||50.00|| |} :- Aliquot 35 + 15ul DNA template :- Mix well. ::''98C for 2min --> [98C for 10sec -> '''60C for 30sec''' -> 72C for 1min] X 8X cycles --> 72C for 2min''<br> :- I load 5ul of amplified DNA at 8 cycles [[File:ZhangLab_2 2014-07-02 21hr 55min_6P_1-10_8x_50ulrxn.jpg|500px]] [[File:ZhangLab_2 2014-07-02 21hr 55min_7P_1-10_8x_50ulrxn.jpg|500px]] * The result looked better than 9 cycles since I can see less background and the border of a smear for samples 6P_1-10 look sharper.Then I continue to do another 50ul PCR using the same condition above. ==== NOTE ==== * Optimization of PCR cycles in small volume (10ul) is still VERY IMPORTANT for RRBS (2-3 cycles different, like 8X cycles and 10X cycles in 2nd round PCR for 1-5ng input) to get a good result even we know roughly cycles # for different input amount. :- I pool ~80ul PCR product from 1st and 2nd times and elute with 55ul EB buffer :- I did PAGE quantification by loading 3ul of bead-purified sequencing libraries <u>'''6-P_1-10: Plasma sample from colon cancer patients'''</u> [[File:ZhangLab_2 2014-07-03 17hr 22min_PQ_6Prrbs_1-10_3ul.jpg| 500px]] <u>'''7-P_1-10: Plasma sample from lung cancer patients'''</u> [[File:ZhangLab_2 2014-07-03 17hr 22min_PQ_7Prrbs_1-10_3ul.jpg| 500px]] ==== PAGE quantification results ==== <u> 006-P samples</u> {| {{table}} border = 1 | align="center" style="width:120px;background:#f0f0f0;"|'''Sample''' | align="center" style="width:80px;background:#f0f0f0;"|'''Index''' | align="center" style="width:80px;background:#f0f0f0;"|'''Conc. (ng/ul)''' | align="center" style="width:80px;background:#f0f0f0;"|'''Amount in 50ul (ng)''' |- | 6-P-1||13||5.18||258.95 |- | 6-P-2||14||4.84||242.15 |- | 6-P-3||15||3.18||159.17 |- | 6-P-4||16||3.82||191.13 |- | 6-P-5||27||2.07||103.50 |- | 6-P-6||18||1.85||92.26 |- | 6-P-7||19||3.04||151.94 |- | 6-P-8||20||2.88||143.84 |- | 6-P-9||21||4.71||235.50 |- | 6-P-10||22||2.59||129.26 |} <u> 007-P samples</u> {| {{table}} border = 1 | align="center" style="width:120px;background:#f0f0f0;"|'''Sample''' | align="center" style="width:80px;background:#f0f0f0;"|'''Index''' | align="center" style="width:80px;background:#f0f0f0;"|'''Conc. (ng/ul)''' | align="center" style="width:80px;background:#f0f0f0;"|'''Amount in 50ul (ng)''' |- | 7-P-1||1||7.11||355.52 |- | 7-P-2||2||5.49||274.61 |- | 7-P-3||4||3.93||196.40 |- | 7-P-4||5||8.14||407.01 |- | 7-P-5||6||3.26||163.24 |- | 7-P-6||7||5.83||291.54 |- | 7-P-7||8||8.10||405.02 |- | 7-P-8||10||9.79||489.42 |- | 7-P-9||11||3.71||185.74 |- | 7-P-10||12||6.48||324.06 |}<br> ==== <u>Library pooling</u> ==== =====<span style="color:mediumblue"><u> 006-P samples</u></span>===== <span style="color:mediumblue">NP-RRBS-6P_1ng-Jun29</span> {| {{table}} border = 1 | align="center" style="width:120px;background:#f0f0f0;"|'''Sample''' | align="center" style="width:60px;background:#f0f0f0;"|'''Index''' | align="center" style="width:80px;background:#f0f0f0;"|'''Conc. (ng/ul)''' | align="center" style="width:80px;background:#f0f0f0;"|'''Volume for 75ng (ul)''' |- | 6-P-1||13||5.18||14.48 |- | 6-P-2||14||4.84||15.49 |- | 6-P-3||15||3.18||23.56 |- | 6-P-4||16||3.82||19.62 |- | 6-P-5||27||2.07||36.23 |- | 6-P-6||18||1.85||40.65 |- | 6-P-7||19||3.04||24.68 |- | 6-P-8||20||2.88||26.07 |- | 6-P-9||21||4.71||15.92 |- | 6-P-10||22||2.59||29.01 |} :- Add 50ul of 6X loading dye and load 73.93 in 4 well of 5-well 6% TBE gel =====<span style="color:red"><u> 007-P samples</u></span>===== <span style="color:red">NP-RRBS-7P_1ng-Jun29</span> {| {{table}} border = 1 | align="center" style="width:120px;background:#f0f0f0;"|'''Sample''' | align="center" style="width:60px;background:#f0f0f0;"|'''Index''' | align="center" style="width:80px;background:#f0f0f0;"|'''Conc. (ng/ul)''' | align="center" style="width:80px;background:#f0f0f0;"|'''Volume for 120ng (ul)''' |- | 7-P-1||1||7.11||16.88 |- | 7-P-2||2||5.49||21.85 |- | 7-P-3||4||3.93||30.55 |- | 7-P-4||5||8.14||14.74 |- | 7-P-5||6||3.26||36.76 |- | 7-P-6||7||5.83||20.58 |- | 7-P-7||8||8.10||14.81 |- | 7-P-8||10||9.79||12.26 |- | 7-P-9||11||3.71||32.30 |- | 7-P-10||12||6.48||18.52 |} :- Add 50ul of 6X loading dye and load 67.31 in 4 well of 5-well 6% TBE gel<br> ---- ==== PAGE size selection ==== * I forgot to take the picture of PAGE size-selection :- NP-RRBS-6P_1ng-Jun29: Resuspend with 12ul H2O x 4tubes --> volume 48ul, 4ul for PAGE verification :- NP-RRBS-7P_1ng-Jun29: Resuspend with 12ul H2O x 4tubes --> volume 48ul, 4ul for PAGE verification ==== PAGE verification ==== === 2014-07-08 === ==== Preparing sequencing library pool for IGM ==== * We will do HiSeq Rapid run, PE 100+7bp * Spike in with 5% PhiX in each lane :1). '''NP_RRBS_PCP-6P_1ng_20140709''' (Loaded in lane 1) :{| {{table}} class = wikitable | align="center" style="width:220px;background:#f0f0f0;"|'''Seq. Library''' | align="center" style="width:60px;background:#f0f0f0;"|'''# of ind. Library''' | align="center" style="width:80px;background:#f0f0f0;"|'''# of read for each (M)''' | align="center" style="width:80px;background:#f0f0f0;"|'''Total # of reads (M)''' | align="center" style="width:60px;background:#f0f0f0;"|'''Loading %''' | align="center" style="width:80px;background:#f0f0f0;"|'''Conc. (nM)''' | align="center" style="width:80px;background:#f0f0f0;"|'''Ratio of % to 6P_1ng''' | align="center" style="width:80px;background:#f0f0f0;"|'''# of mole to pool (nmole)''' | align="center" style="width:80px;background:#f0f0f0;"|'''Volume to pool (ul)''' |- | NP-RRBS-6P_1ng-Jun29||10||10||100||66.67||13.79||1||120||8.70 |- | NP-RRBS-PC_P_1ng_p1-Jun20||8||5||40||26.67||28.25||0.4||48||1.70 |- | NP-RRBS-PC_P_1ng_p2-Jun20||2||5||10||6.67||15.90||0.1||12||0.75 |- | ||||||||||||Total||180||11.16 |- | ||||||||||||Final conc.||16.14||nM |} :2). '''NP_RRBS_NCP-7P_1ng_20140709''' (Loaded in lane 2) :{| {{table}} class = wikitable | align="center" style="width:220px;background:#f0f0f0;"|'''Seq. Library''' | align="center" style="width:60px;background:#f0f0f0;"|'''# of ind. Library''' | align="center" style="width:80px;background:#f0f0f0;"|'''# of read for each (M)''' | align="center" style="width:80px;background:#f0f0f0;"|'''Total # of reads (M)''' | align="center" style="width:60px;background:#f0f0f0;"|'''Loading %''' | align="center" style="width:80px;background:#f0f0f0;"|'''Conc. (nM)''' | align="center" style="width:80px;background:#f0f0f0;"|'''Ratio of % to 7P_1ng''' | align="center" style="width:80px;background:#f0f0f0;"|'''# of mole to pool (nmole)''' | align="center" style="width:80px;background:#f0f0f0;"|'''Volume to pool (ul)''' |- | NP-RRBS-7P_1ng-Jun29||10||10||100||71.43||16.89||1||120||7.10 |- | NP-RRBS-NC_P_1ng_p1-Jun20||4||5||20||14.29||18.95||0.2||24||1.27 |- | NP-RRBS-NC_P_1ng_p2-Jun20||4||5||20||14.29||15.80||0.2||24||1.52 |- | ||||||||||||Total||168||9.89 |- | ||||||||||||Final conc.||16.99||nM |}
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