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= scRBS experiment of single mouse neuronal nucleus (Exp #2) = [[noi:DMR220k_LabNotes#2014|'''Back to calendar''']]<br> * This experiment continued from the the first of experiment (Exp#1) for UCLA (Dr. Fan's group) * I had some discussion in previous experiment. For this experiment, I will MONITOR PCR in the 1st PCR carefully to prevent over-amplification of adaptor dimers. * In addition, I will include more control either positive control and negative control in every step to make sure that where the signal comes from. I will also include and exclude unmeth lambda DNA as well. == Sample list in each step in brief == ==== Cell lysis ==== :- WT_1 to WT_16 (WT 1 nu, performed by Youjin). I re-run the number of samples to make it easy to handle and track. :For my part I will include all controls and also in the following steps :- T_1 to T_16 (TKO 1 nu, performed by Noi). :- T10, T100 and T0 (TKO 10nu, 100nu and 0nu, 1 each, performed by Noi) :- 10pg of CTT with unmeth lambda DNA (duplicates). CTT is gDNA from frozen colon tumor tissue purchased from Biochain. :- NTC with unmeth lambda DNA (duplicate), * All samples in this step will have 60fg of unmeth lambda DNA spike in. This allows for bisulfite conversion efficiency measurement. :- I did serial dilution of CTT gDNA from 2ng/ul to 100pg/ul and 5pg/ul ::- Mix 10ul of 2ng/ul CTT gDNA with 190ul H2O ::- Mix 10ul of 100pg/ul CTT gDNA with 190ul H2O ==== DNA fragmentation with MspI ==== :- 10pg of CTT '''without''' unmeth lambda DNA (duplicates). :- MspI NTC '''without''' unmeth lambda DNA (duplicates). ==== Methylated adaptor ligation ==== :- Ligation control (duplicates). * No NTC during Gap-filling/dA-tailing == Experimental procedures == * I used mouse neuronal nuclei picked on 2014-12-17 and stored at -80C === 1) Cell lysis === '''''<span style="color:crimson"><u>Prep</u></span>'''''<br> - Thaw nuclei from -80C & spin down at 2,000rpm for 5min. I put PCR tube on PCR rack. (96-well plate rotor, 5min)<br> - Mix QIAGEN Protease with lambda DNA. I prepared more than enough of protease and unmethy lambda DNA mix. :{| {{table}} border = 1 | align="center" style="width:180px;background:#f0f0f0;"|'''Components''' | align="center" style="width:80px;background:#f0f0f0;"|'''1X rxn''' | align="center" style="width:80px;background:#f0f0f0;"|'''100X rxn''' |- | Cell in 1.43X lysis buffer||4.00||0.00 |- | 1.2pg Lambda DNA||0.05||5.00 |- | Protease||1.00||100.00 |- | Total||5.05|| |} * Final amount of unmet lambda DNA in each tube is 60fg. :- Add 1.05 ul of protease and unmeth lambda DNA mix to each tube. No need to pipette! :- Spin down the tube on PCR rack 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 spin down the plate before and after mixing quite long to make sure that all reagents were collected to the bottom of the tube :- ''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 * Note that before cell lysis and after cell lysis before MspI digestion, I need to spin the tube containing single nucleus at high speed and long time to make sure that I do not lose nucleus. <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}} border = 1 | align="center" style="width:180px;background:#f0f0f0;"|'''Components''' | align="center" style="width:80px;background:#f0f0f0;"|'''Volume (ul)''' | align="center" style="width:80px;background:#f0f0f0;"|'''42x rxn mix''' |- | Lysed nuclei||5.00||0.00 |- | 10X Tango buffer||2.00||84.00 |- | MspI||0.90||37.80 |- | H2O||10.10||424.20 |- | Total||18.00|| |} :- Set up positive control of 10pg CTT gDNA (no lambda DNA) and MspI NTC (no lambda DNA) in duplicates ::- 10pg CTT: Add 2ul of 5pg/ul CTT gDNA and 3ul H2O ::- MspI NTC, Add 5ul H2O :- Aliqout 13ul of MspI reaction mix to each tube :- 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'' :- Set program to hold at 4C :- Spin down the plate at 2,000rpm for 1min before continuing to next step <br> === 3) End-repair or 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> :- Mix 50 dA:dC:dG mix (20mM:2mM:2mM) with 35ul of Klenow fragment exo- (just before adding to MspI-digested DNA) :- Add 2ul of dA:dC:dG/Klenow fragment exo- mix to each tube. Do not need to touch the reaction. Just touch the tip of pipette tip close enough to the reaction and deliver reaction 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 :- ''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 1:25 diluted Illumina indexed methylated adaptor (TruSeq adaptor) in total reaction 25ul at 16C for 30min and 4C for at least 16h. * Final conc. of adaptor in 25ul ligation reaction is ~23nM ** In Dr. Tang's paper, he used 1ul of 1:20 diluted TruSeq adaptor (final conc. ~28.5nM * In this experiment, I made fresh batch of 1:25 diluted TruSeq adaptor in 0.2mL 8-tube strip by mixing 2ul of 15uM of TruSeq adaptor with 50ul H2O :{| {{table}} border = 1 | align="center" style="width:180px;background:#f0f0f0;"|'''Components''' | align="center" style="width:80px;background:#f0f0f0;"|'''Volume (ul)''' | align="center" style="width:80px;background:#f0f0f0;"|'''50x rxn mix''' |- | dA-tailed reaction||20.00||0.00 |- | 10X Tango buffer||0.50||25.00 |- | HC T4 DNA ligase (30units/ul)||1.00||50.00 |- | 10mM ATP||1.25||62.50 |- | H2O||1.25||62.50 |- | Total||24.00|| |} :- Add 1ul of 1:25 diluted TruSeq adaptor to each well with multichannel pipette :- Add 4ul of ligation reaction mix to each tube :- 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 30min and 4C O/N (~20h, no heat lid)'' :- ''Heat inactivate at 65C for 20min''. This time I skipped this step :- Spin down the plate at 2,000rpm for 1min before continuing to next step * Note: The way I assigned TruSeq index was based on Youjin suggestion. The reason was that we had limited number of indexed barcode (24), and Youjin wanted to combined both WT and TKO for sequencing in the same lane. This is why I used duplicated indexes for sample in the same group (WT or TKO) and have at least 12 different indexes between the two groups (Index 1-12 for WT and Index 13-27 for TKO, no index #17, 24, and 26. :{| {{table}} class = wikitable | align="center" style="width:80px;background:dodgerblue;"|'''Sample ID''' | align="center" style="width:80px;background:magenta;"|'''TruSeq Index#''' | align="center" style="width:80px;background:springgreen;"|'''Seq(RC)''' | align="center" style="width:80px;background:dodgerblue;"|'''Sample ID''' | align="center" style="width:80px;background:magenta;"|'''TruSeq Index#''' | align="center" style="width:80px;background:springgreen;"|'''Seq(RC)''' |- | WT_1||1||ATCACG||T_1||13||AGTCAA |- | WT_2||2||CGATGT||T_2||14||AGTTCC |- | WT_3||3||TTAGGC||T_3||15||ATGTCA |- | WT_4||4||TGACCA||T_4||16||CCGTCC |- | WT_5||5||ACAGTG||T_5||18||GTCCGC |- | WT_6||6||GCCAAT||T_6||19||GTGAAA |- | WT_7||7||CAGATC||T_7||20||GTGGCC |- | WT_8||8||ACTTGA||T_8||21||GTTTCG |- | WT_9||9||GATCAG||T_9||22||CGTACG |- | WT_10||10||TAGCTT||T_10||23||GAGTGG |- | WT_11||11||GGCTAC||T_11||25||ACTGAT |- | WT_12||12||CTTGTA||T_12||27||ATTCCT |- | WT_13||1||ATCACG||T_13||13||AGTCAA |- | WT_14||2||CGATGT||T_14||14||AGTTCC |- | WT_15||3||TTAGGC||T_15||15||ATGTCA |- | WT_16||4||TGACCA||T_16||16||CCGTCC |} :{| {{table}} class = wikitable | align="center" style="width:80px;background:dodgerblue;"|'''Sample ID''' | align="center" style="width:80px;background:magenta;"|'''TruSeq Index#''' | align="center" style="width:80px;background:springgreen;"|'''Seq(RC)''' | align="center" style="width:80px;background:dodgerblue;"|'''Sample ID''' | align="center" style="width:80px;background:magenta;"|'''TruSeq Index#''' | align="center" style="width:80px;background:springgreen;"|'''Seq(RC)''' |- | T10nu||18||GTCCGC||CTT-nolambda-1||9||GATCAG |- | T100nu||19||GTGAAA||CTT-nolambda-2||10||TAGCTT |- | T0nu||20||GTGGCC||MspI-NTC-nolambda-1||11||GGCTAC |- | CTT+lambda-1||21||GTTTCG||MspI-NTC-nolambda-2||12||CTTGTA |- | CTT+lambda-2||22||CGTACG||Lig-NTC-1||13||AGTCAA |- | NTC+lambda-1||23||GAGTGG||Lig-NTC-2||14||AGTTCC |- | NTC+lambda-2||25||ACTGAT|||||| |} === 6) Bisulfite conversion === * I performed bisulfite conversion using MethylCode™ Bisulfite Conversion Kit from LifeTechnologies and used the same procedure following manufacturer's instruction and elute with 32ul elution buffer. * Note: Zymo EZ DNA Methylation-Gold™ Kit is identical to MethylCode™ Bisulfite Conversion Kit '''''<span style="color:crimson"><u>Prep</u></span>'''''<br> - Prepare 5 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 (This time, I diluted a new tube of 10ng/ul yeast tRNA from stock 1ug/ul)<br> - For 45.5 rxn, I mixed 27.30mL of Binding Buffer with 45.5 ul of 10ng/ul tRNA in 50mL tube. : - 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. :- 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) 32ul 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 === 7) PCR amplification === ==== <u>1st round PCR</u> ==== '''''<span style="color:crimson"><u>Prep</u></span>'''''<br> * I used all eluted bisDNA in the 1st round PCR :{| {{table}} border = 1 | align="center" style="background:#f0f0f0;"|'''Components''' | align="center" style="width:80px;background:#f0f0f0;"|'''Conc''' | align="center" style="width:80px;background:#f0f0f0;"|'''unit''' | align="center" style="width:80px;background:#f0f0f0;"|'''Final conc.''' | align="center" style="width:80px;background:#f0f0f0;"|'''unit''' | align="center" style="width:80px;width:80px;background:#f0f0f0;"|'''Volume (ul)''' | align="center" style="width:80px;background:#f0f0f0;"|'''50 rxn mix''' |- | Bis-cvt DNA||||||||||30.00||0.00 |- | 10X Reaction buffer||10||X||1||X||5.00||250.00 |- | dNTP mix||10||mM||0.3||mM||1.50||75.00 |- | TruF/R||10||uM||0.3||uM||1.50||75.00 |- | PfuTurbo Cx||2.5||Unit/ul||1||unit||0.40||20.00 |- | 50X SYBG||50||X||0.4||X||0.40||20.00 |- | H2O||||||||||11.20||560.00 |- | Total||||||||||50.00|| |} :- Aliquot 20ul, add 30ul of bisulfite-treated adaptor-ligated DNA ::''95C for 2min --> [95C for 20sec -> '''60C for 30sec''' -> 72C for 1min] X 24 cycles --> 72C for 2min''<br> * I stopped 1st round PCR at 17 cycles as the qPCR curve was close to saturate. * I was still worried about the cycle number as 17 cycles was much less than 25 cycles by Tang's group. However, I could not let it over amplified of adaptor dimers as well. ::- I took 5ul of 1st PCR product of all samples for PAGE verification. * This might take a lot of time and TBE gel to verify. However, I want to observe the result in every single step to be able to interpret the result and get optimum condition for scRRBS. ==== 1st PCR at 17 cycles, 5ul PCR product ==== [[File:ZhangLab_2 2014-12-22 14hr 53min_1stPCR-gel1.jpg| 450px]]<br> <br> [[File:ZhangLab_2 2014-12-22 14hr 53min_1stPCR-gel2.jpg| 450px]]<br> <br> [[File:ZhangLab_2 2014-12-22 14hr 53min_1stPCR-gel3.jpg| 450px]]<br> * All samples in 3 images above (W_1 to W_16 and T_1 to T_16) had 60fg of unmeth lambda DNA spike in [[File:ZhangLab_2 2014-12-22 14hr 53min_1stPCR-gel4.jpg|350px]]<br> <br> [[File:ZhangLab_2 2014-12-22 14hr 53min_1stPCR-gel5.jpg| 320px]] * NTC_1 and NTC_2 are NTC during cell lysis step. * Idid not see any different between 1nu reaction (W_XX and T_XX) and NTC in cell lysis, MspI and ligation control. Most of the signals came from primers, adaptors and adaptor dimers and faint band below 200bp band. * However, for reaction with 10 and 100nuclei (T10n and T100n) I noticed a faint smear below and above 200bp as cropped in image. In addition, the intensity of smear was proportional to number of nuclei in the reaction. I was hoping that it is the real signal of RRBS libraries from mouse nuclei. * The next challenging for single nuclei reaction was how to remove the background of adaptor dimers ~125bp which was a dominant background and the size close to the size of expected libraries and increase amplification of the real signal which was not visible in TBE gel. * I continued to purify the 1st round amplicons with AMPure beads twice (1:1 ratio). Left over PCR product volume was 45 ul. ==== <u>AMPure bead purification</u> ==== '''''<span style="color:crimson"><u>Prep</u></span>'''''<br> - Freshly prepare 80% EtOH :- Add 45ul AMPure bead. Mix by pipetting 10x :- Sit for 8min :- Transfer to sit on magnet for 5min :- Wash twice with 200ul 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 200ul 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> ==== '''''<span style="color:crimson"><u>Prep</u></span>'''''<br> * I did qPCR in small volume (10ul in duplicates) to monitor PCR cycle number. :{| {{table}} border = 1 | align="center" style="background:#f0f0f0;"|'''Components''' | align="center" style="width:80px;background:#f0f0f0;"|'''Conc''' | align="center" style="width:80px;background:#f0f0f0;"|'''unit''' | align="center" style="width:80px;background:#f0f0f0;"|'''Final conc.''' | align="center" style="width:80px;background:#f0f0f0;"|'''unit''' | align="center" style="width:80px;background:#f0f0f0;"|'''Volume (ul)''' | align="center" style="width:80px;background:#f0f0f0;"|'''50 rxn mix''' |- | Purified 1st round DNA||||||||||6.00||0.00 |- | 5X Phusion HF buffer||5||X||1||X||4.00||200.00 |- | dNTP mix||10||mM||0.3||mM||0.60||30.00 |- | TruS_F/R||10||uM||0.3||uM||0.60||30.00 |- | 50X SYBR||50||X||0.4||X||0.16||8.00 |- | Phusion HF||2||unit/ul||||||0.20||10.00 |- | H2O||||||||||8.44||422.00 |} :- Aliquot 14ul + 6ul of template ::''98C for 2min --> [98C for 10sec -> '''60C for 30sec''' -> 72C for 1min] X XX cycles --> 72C for 2min''<br> ::- I stopped 2nd PCR af different cycle number based on qPCR curve. Do not let it saturated. :- Strip 1: W_1 to W_8 :- Strip 2: W_9 to W_16 :- Strip 3: T_1 to T_8 :- Strip 4: T_9 to T_16 :- Strip 5: T10n, T100n, T0n, CTT+lamb_1, CTT+lamb_2, NTC+lamb_1, NTC+lamb_2 :- Strip 6: CTT-lamb_1, CTT-lamb_2, MspI-lamb_NTC_1, MspI-lamb_NTC_2, Lig-lamb_NTC_1, Lig-lamb_NTC_2 * I stopped reaction at different cycle number :- Strip 1-4, 6 stopped at 9 and 11 cycles :- Strip 5: stopped at 8 and 9 cycles. :- I verified 5ul of PCR product in 6% TBE gel. [[File:ZhangLab_2 2014-12-22 18hr 25min_2ndPCR-gel1.jpg| 450px]]<br> <br> [[File:ZhangLab_2 2014-12-22 18hr 25min_2ndPCR-gel2.jpg| 450px]]<br> <br> [[File:ZhangLab_2 2014-12-22 18hr 25min_2ndPCR-gel3.jpg| 450px]]<br> <br> * All samples in 3 images above (W_1 to W_16 and T_1 to T_16) had 60fg of unmeth lambda DNA spike in [[File:ZhangLab_2 2014-12-22 18hr 25min_2ndPCR-gel5.jpg| 400px]]<br> <br> [[File:ZhangLab_2 2014-12-22 18hr 25min_2ndPCR-gel6.jpg| 400px]]<br> <br> [[File:ZhangLab_2 2014-12-22 18hr 25min_2ndPCR-gel4.jpg| 380px]]<br> <br> * To confirm that the intensity of reaction with 1 nucleus was higher than NTC_1 and NTC_2 (with lambda DNA spike in), I loaded some samples of wild type (W_1 to W_5) and TKO (T_1 to T_4) at 9 cycles. I did not have NTC_1 and NTC_2 with lambda DNA spike in at 11 cycles for comparison. [[File:ZhangLab_2 2014-12-22 18hr 25min_2ndPCR-gel7_verify-2NTC-5WT-4TKO-at9cycles.jpg| 500px]]<br>
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