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* Update 10/6/2015 - failed to generate libraries. = 1.5 ng RRBS libraries from plasma DNA = * Experiments will be performed in two batches. I randomly split the samples into two batches as follows. The tube IDs will correspond to the TruSeq Barcode ids as well (with exception of Index 17 will be Index 27 because we dont have Index 17). * Each batch will have 2 NTCs, and 2 controls (1 ng and 2 ng from the combined tube.) {| class="wikitable" | align="center" style="background:#f0f0f0;"|'''Batch number''' | align="center" style="background:#f0f0f0;"|'''Tube ID/TruSeq ID''' | align="center" style="background:#f0f0f0;"|'''Sample ID''' | align="center" style="background:#f0f0f0;"|'''Volume for 1.5 ng (uL)''' | align="center" style="background:#f0f0f0;"|'''Volume water to make 14.17 uL (uL)''' |- | 0||1||6.11||6.10||8.07 |- | 0||2||6.16||3.83||10.34 |- | 0||3||6.17||5.64||8.53 |- | 0||4||6.18||4.75||9.42 |- | 0||5||6.22||1.16||13.01 |- | 0||6||6.23||1.71||12.46 |- | 0||7||6.24||4.10||10.07 |- | 0||8||6.25||6.47||7.70 |- | 0||9||6.26||4.57||9.60 |- | 0||10||6.27||8.82||5.35 |- | 0||11||7.11||3.55||10.62 |- | 0||12||7.14||4.05||10.12 |- | 0||13||7.15||4.66||9.51 |- | 0||14||7.19||3.41||10.76 |- | 0||15||7.22||1.95||12.22 |- | 0||16||7.23||1.25||12.92 |- | 0||27||7.24||0.01||14.16 |- | 0||18||7.25||5.14||9.03 |- | 0||19||7.26||5.68||8.49 |- | 0||20||7.29||5.43||8.74 |- | 1||1||6.12||4.81||9.36 |- | 1||2||6.13||1.44||12.73 |- | 1||3||6.14||3.15||11.02 |- | 1||4||6.15||7.73||6.44 |- | 1||5||6.19||3.81||10.36 |- | 1||6||6.20||8.33||5.84 |- | 1||7||6.21||3.95||10.22 |- | 1||8||6.28||0.37||13.80 |- | 1||9||6.29||4.78||9.39 |- | 1||10||6.30||3.50||10.67 |- | 1||11||7.12||9.62||4.55 |- | 1||12||7.13||4.66||9.51 |- | 1||13||7.16||5.10||9.07 |- | 1||14||7.17||1.38||12.79 |- | 1||15||7.18||2.12||12.05 |- | 1||16||7.20||2.01||12.16 |- | 1||27||7.21||4.08||10.09 |- | 1||18||7.27||2.73||11.44 |- | 1||19||7.28||1.42||12.75 |- | 1||20||7.30||0.65||13.52 |} === Prepare labeled strip tubes and combine DNA with water to make 14.17 total volume === === Msp I digestion === * To 14.17 uL volume of DNA and nuclease free water add 3.83 uL of enzyme, buffer, and lambda control DNA. * Master mix, make 25X: <nowiki>MspI (10U/uL) 1.00 uL 10x Tango Buffer 2.00 uL 12pg/ul unmeth lambda DNA 0.83 uL </nowiki> *Spin down the tubes, mix by gentle pulse vortexing on PCR 10x *Spin down the tubes, *Incubate 37C O/N (~13 hr) β 65C 20 min β hold at 4C ===End-repair/dA-tailing=== * Master mix, make 25X: <nowiki>MspI digested DNA 18.00 uL Klenow exo- 1.00 uL dATP:dCTP:dGTP (20mM,2mM,2mM) 1.00 uL</nowiki> *Spin down the tubes, mix by gentle pulse vortexing on PCR rack 10x *Spin down the tubes, *Incubate 30C 20 min β 37C 20 min β hold at 4C (no heated lid) *Heat inactive 75C 10 min β 4C (with heated lid) ===Methylated adaptor ligation=== <nowiki>dA-tailed DNA 20.00 uL 10X Tango Buffer 0.50 uL HC T4 DNA ligase(30U/uL) 1.00 uL 10mM ATP 1.25 uL Nuclease free water 1.25 uL 1:20 diluted TruSeq adaptors 1.00 uL</nowiki> *Spin down the tubes, mix by gently pulse vortexing on PCR rack 10x *Spin down the tubes, *Incubate 16C, 22 h (no heated lid, >=20 hr) *Mix by gentle pulse vortexing on PCR rack 5x *Spin down *Heat inactivate at 65C, 20 min β hold 4C ===AMPure beads purification=== * Add 5 uL of water to make total volume equals 30 uL of ligation mixture. * Add 60 uL of AMPure beads and repipette 10X to mix. * Incubate for 30 minutes at room temperature. * Wash twice with freshly prepared 70% ethanol. * Dry for 15 minutes at room temperature. (While drying, label and UV new tubes for bisulfite conversion below). * Add 30 uL of EB buffer to elute. ===Bisulfite conversion using LifeTech MethylCode Bisulfite conversion kit=== * Prepare 3 tubes of complete CT Conversion Reagent by adding 800ul 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 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 98Β°C for 10 minutes (DNA denaturation) 64Β°C for 2.5 hours (Bisulfite conversion) 4Β°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 * Discard spnt * Add 100ul Wash Buffer (Aliquot Wash Buffer in 5mL tube) * Spin down column at 14,000 rpm for 30s (do not discard spnt) * Add 200ul Desulfonation Buffer (aliquot Desulfonation Buffer in 2mL or 5mL tube). Incubate for 15-20 minutes at room temperature. * Spin down column at 14,000 rpm for 30s (do not discard spnt) * Add 200ul Wash Buffer * Spin down column at 14,000 rpm for 30s * Discard spnt * Add 200ul Wash Buffer * Spin down column at 14,000 rpm for 3min (do not discard spnt) * 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 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 === * Perform amplification without Sybr. * Perform PCR with 12 cycles (fixed), 50 uL total volume. <nowiki> bis-cvt DNA 30.00 uL 10X Reaction buffer 5.00 uL dNTP, 10mM 1.25 uL TruSeq F/R, 10uM 1.50 uL PfuTurboCX 1.00 uL Nuclease free water 11.25 uL </nowiki> *Mix well *Program: 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 15 minutes, wash twice with freshly prepared 80% ethanol, dry beads for 20 minutes (?)) *Elute with 20 uL EB buffer *Continue to 2nd round PCR. No PAGE Verification * Test 2nd round PCR * Noi's protocol: <nowiki> Purified 1st round DNA 3.00 uL 2X Phusion HS Flex MM 5.00 uL TruSeq F/R, 10 uM 0.30 uL Nuclease free water 1.70 uL </nowiki> *Mix well *Program: 98C for 2min β [98C for 10sec β 60C for 30sec β 72C for 1min] X 9X cycles β 72C for 2min *Load everything to 6% gel and run at 225 V for 30 min. * Once PCR cycle is determined (8-9 cycles?), prepare PCR for all the samples and then purify with AMPure beads. Run gel to also determine the pooling ratios and perform size selection for 150-400 bp bands.
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