Noi/NOTES/2014-12-17
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scRBS experiment of single mouse neuronal nucleus (Exp #1)[edit]
- This experiment, I will generate RRBS libraries of mouse single nuclei, including control:wild-type; and TKO: Dnmt1; 3a; 3b. Youjin from UCLA will pick single nuclei under microscope for this experiment. The method for cell picking would be different from previous samples I got from Scripp.
- NOTE that I have performed two experiments, including this experiment and one on 2014-12-20. The first experiment (this experiment) seemed to fail and difficult to interpret the result. However, I would document all details in each experiment for comparison the conditions with the one that we think it worked well.
Experimental procedures[edit]
1) Cell picking[edit]
- Youjin preferred to have lysis buffer (as bedding buffer) in 0.2mL PCR tube as it's convenient for him to handle one tube at a time and to avoid contamination.
- He said that the volume of picked nucleus and buffer should be ~0.5ul, so I prepare 3.5ul of 1.43 x Lysis buffer to get a final concentration of 1x after loading 0.5ul of picked nucleus and 1ul of protease.
- Since cell picking finished pretty late, so I stored picked nuclei in -80C and continued cell lysis on the following day.
2) Cell lysis[edit]
Prep
- Thaw nuclei from -80C & spin down at 2,000rpm for 5min. I put PCR tube on PCR rack. (96-well plate rotor, 5min)
- Mix QIAGEN Protease with lambda DNA. I prepared more than enough of protease and unmethy lambda DNA mix.
Components 1X rxn 70X rxn Cell in 1.43X lysis buffer 4.00 0.00 1.2pg Lambda DNA 0.05 3.50 Protease 1.00 70.00 Total 5.05
- Final amount of unmet lambda DNA in each tube is 60fg.
Sample list[edit]
- WT = wild type
- T= TKO
- Number after WT or T like W10, T100 is numb rod nuclei the rest are W or T with single nucleus and running order number.
WT_1 WT_2 WT_3 WT_4 WT_5 WT_6 WT_7 WT_8 WT_9 WT_10 WT_11 WT_12 T_1 T_2 T_3 T_4 T_5 T_6 WT10_1 WT10_2 WT100 0nu NTC T_7 T_8 T_9 T_10 T_11 T_12 T10 T100 NTC 0nu
- I handled sample WT-1 --> NTC (23 samples)
- Youjin handled sample T7 --> 0nu (10 samples)
- I prepared the same master mix then aliquot for Youjin for every step.
- Note that Youjin did not pick 0 nuclei from the nucleus suspension which is ideal negative control to make sure that the signals do not come from buffer or other sources. We therefore use lysis buffer that I aliquot as bedding buffer as 0nu control.
- For NTC, I aliquot lysis buffer from original tube of lysis buffer and add H2O instead of pick nuclei.
- - 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
- - 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 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.
3) DNA fragmentation with MspI[edit]
- Incubated released naked DNA with 9units of MspI in 18ul reaction at 37C for 3hr.
Prep
- Prepare MspI reaction mix
Components | Volume (ul) | 34x rxn mix |
Lysed nuclei | 5.00 | 0.00 |
10X Tango buffer | 2.00 | 68.00 |
MspI | 0.90 | 30.60 |
H2O | 10.10 | 343.40 |
Total | 18.00 |
- - Add 13ul to each well with multi-channel pipette
- - 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
4) End-repair or gap-filling/dA-tailing[edit]
- 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
Prep
- - Mix 50 dA:dC:dG mix (20mM:2mM:2mM) with 50ul 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
5) Methylated adaptor ligation[edit]
- 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.
- In Dr. Tang's paper, he used 1ul of 1:20 diluted TruSeq adaptor
- We have modified to incubate at 16C O/N
- For this round of experiment, Youjin preferred to use STD Illumina adaptor as he wanted to include all samples in the same lane for sequencing in case that the experiment works well.
Components | Volume (ul) | 36x rxn mix |
dA-tailed reaction | 20.00 | 0.00 |
10X Tango buffer | 0.50 | 18.00 |
HC T4 DNA ligase (30units/ul) | 1.00 | 36.00 |
10mM ATP | 1.25 | 45.00 |
15uM Univ adaptor | 0.02 | 0.72 |
H2O | 2.23 | 80.28 |
Total | 25.00 |
- - Add 5ul of ligation reaction mix with 12nM of STD Illumina adaptor
- - 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 O/N (~18h, 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
6) Bisulfite conversion[edit]
- I performed bisulfite conversion using MethylCode™ Bisulfite Conversion Kitfrom LifeTechnologies and used the same procedure following manufacturer's instruction and elute with 32ul elution buffer.
Prep
- Prepare 4 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
Prep
- Mix 600:1 ratio of Binding Buffer and 10ng/ul tRNA
- For 33.5 rxn, I mixed 20.10mL of Binding Buffer with 33.5 ul of 10ng/ul tRNA
- - 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. Discard spnt
- - 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. Discard spnt
- - 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[edit]
1st round PCR[edit]
Prep
Components Conc unit Final conc. unit Volume (ul) 36 rxn mix Bis-cvt DNA 30.00 0.00 10X Reaction buffer 10 X 1 X 5.00 180.00 dNTP mix 10 mM 0.2 mM 1.00 36.00 PCR_F 10 uM 0.3 uM 1.50 54.00 PCR_R.IndX 5 uM 0.3 uM 3.00 108.00 PfuTurbo Cx 2.5 Unit/ul 1 unit 0.40 14.40 50X SYBG 50 X 0.4 X 0.40 14.40 H2O 8.70 313.20
- - Aliquot 17ul, add 3ul of 5uM PCR_R.IndX, 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
- 95C for 2min --> [95C for 20sec -> 60C for 30sec -> 72C for 1min] X 24 cycles --> 72C for 2min
- The qPCR curve was saturated as it reach ~18-19 cycles
- - I took 5ul of 1st PCR product (sample WT_1-WT_8) for PAGE verification,
File:ZhangLab 2 2014-12-20 10hr 42min 1stPCR-5ul 1-8-of1nu-NP.jpg
- - Purified the 1st round amplicons with AMPure beads 2X (1:1 ratio)
AMPure bead purification[edit]
Prep
- Freshly prepare 80% EtOH
- - Add 50ul 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 (make sure that the beads are completely dried out to avoid EtOH inhibiting PCR)
- - 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
2nd round PCR[edit]
Prep
Components Conc unit Final conc. unit Volume (ul) 35 rxn mix Purified 1st round DNA 20.00 0.00 5X Phusion HF buffer (CG) 5 X 1 X 10.00 350.00 dNTP mix 10 mM 0.2 mM 1.00 35.00 PCR_F 10 uM 0.3 uM 1.50 52.50 PCR_R Index 5 uM 0.3 3.00 0.00 50X SYBR 50 X 0.4 X 0.40 14.00 Phusion HF 2 unit/ul 0.50 17.50 H2O 13.60 476.00
- - Aliquot 27ul + 3ul of PCR_R Ind primer + 20ul template
- - I reduced amount of 1st PCR template in 2nd PCR
- 98C for 2min --> [98C for 10sec -> 60C for 30sec -> 72C for 1min] X 6 cycles --> 72C for 2min
- - I stopped 2nd PCR very early as the qPCR curve getting saturated.
- - I verified PCR product in 6% TBE gel
File:ZhangLab 2 2014-12-20 16hr 07min 2ndPCR-6cycle-NP-gel1.jpg File:ZhangLab 2 2014-12-20 16hr 07min 2ndPCR-6cycle-NP-gel2.jpg File:ZhangLab 2 2014-12-20 16hr 07min 2ndPCR-6cycle-NP-gel3.jpg File:ZhangLab 2 2014-12-20 16hr 06min 2ndPCR-6cycle-YH-lower-PQ.jpg
Discussion[edit]
- From the result, I could not distinguish the reactions between 1nu and 0nu as I saw a very strong smear along the lane.
- I saw stronger signal in 10nu and 100nu. However the background was still very high and the pattern looked different from RRBS sequencing libraries. In addition, the range of sequencing libraries was too board to select for sequencing.
- Youjin noticed strong signal at ~200bp or below in some samples, and he was interested to do sequencing on those samples that has been cropped in the gel images. However, for me, it's very difficult to interpret the result. I
What I have learnt from this experiment[edit]
- When first PCR was allowed to saturate, it generate very strong background a long the lane in all samples. This background might come from adaptors.
- After 2 round of bead purification, it can remove only the signal below 150bp or so. However the smear along the lane is still there. This background will interfere 2nd PCR, so we would see it getting saturated at very early cycle number (6cycles). These background overcomes the real signal of library from the cell.
- To resolve this, I plan to repeat experiment and tried to follow what I have done before like using TruSeq adaptor. This make me easy when set up PCR and follow incubation condition for ligation reaction.
- The most important thing I would do is that I will monitor qPCR of 1st PCR and stopped once get close to the plateau. This will prevent the background from over amplified of adaptor dimers. This may be risky if the PCR cycle number is too small to amplify enough signal of the real template. I could lose a lot of PCR product of the real signal by performing 2 rounds of bead purification.
- I would not say that this experiment totally failed, but the sub optimized condition of PCR cycle number in 1st PCR that made the background was too high.