Noi/NOTES/2014-2-17

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scRRBS trial experiment #3[edit]

  • Link to calendar: [[1]]
  • Continued from: [[2]]

FACTORS HAVE BEEN CHANGED[edit]

  1. Included only 0, 10, 100 1000 nuclei since 1 nuclei was not different from 0 nuclei. I wanted to get the condition that work well for 10 nuclei which is the minimum nuclei number that close to 1 nuclei.
  2. Reduce amount of methylated adapter
    1. For 0 and 10 nuclei: 100 time less from previous experimentt (equal to adapter for 10pg input, based on 10:1 ratio)
    2. For 100 and 1000 nuclei: 10 times less from previous experiment (equal to adapter for 100pg, based on 10:1 ratio)
  3. TEST the effect of PEG 4000 for ligation reaction
    1. - PEG 4000
    2. + PEG 4000
  4. For gap-filling/dA-tailing, incubate 30C 20min, 37C for 10min for optimal end-repair and dA-tailing, respectively
  5. Heat inactivate Klenow fragment exo- after gap-filling/dA-tailing
  6. TEST incubation time during ligation
    1. 22C for 1h as recommended from Thermoscientific
    2. 16C for 16h-20h as recommended from Gu H et al, 2011 that ligation time is critical for low input RRBS, especially for input < 200ng. I plan to incubate for 16h.
  7. Reduce primer in 1st round of amplification to 0.1uM
  8. I tried not to mix reaction by pipetting since I don't want to lose DNA to the pipette tip. I only add reagent close to the top of reaction, spin down, vortex gently (avoid generating bubble), and spin down again.

Experimental procedures[edit]

Layout[edit]
Well #1 Well #2 Well #3 Well #4
NTC 10 nuclei 100 nuclei 1000 nuclei
  • Four replicates were perform to test the effect of PEG and incubation time
    • Group 1: -PEG, 22C for 1h
    • Group 2: -PEG, 16C for 16h
    • Group 3: +PEG, 22C for 1h
    • Group 4: +PEG, 16C for 16h

Cell lysis[edit]

- Thaw nuclei from -20C & spin down
- Add 0.5ul of protease
- Incubate at 50C for 3hr
- Heat inactivate at 75C for 30min
  • NOTE: I did not include unmethylated lambda DNA in this experiment since I think that I only test the condition. Next time I should include lambda DNA in case that the experiment works well, I could continue to sequencing.


Msp I digestion[edit]

Components Volume (ul) 17x rxn mix
Lysed nuclei 5.00 0.00
10X Tango buffer 2.00 34.00
MspI 0.90 15.30
H2O 10.10 171.70
Total 18.00 221.00
- Aliquot 13ul to each tube (no mixing by pipetting)
- Spin down -> vortex gently ->spin down -> vortex gently -> spin down
- Incubate at 37C for 3hr

End-repair/dA-tailing[edit]

- Add 1ul of dA/dC/dG mix
- Add 1ul of Klenow fragment exo-
- Incubate at 30C for 15min
- Heat inactivate at 75C for 10 min**


Methylated adapter ligation[edit]

- Prepare new tube of methylated adapter
- Dilute 2ul of stock concentration in 1000ul --> for 1ng input
- Dilute 1ng input to 100pg input (100ul 1ng input + 900ul H2O)
- Dilute 1ng input tp 10pg input (10ul 1ng input + 990ul H2O)

- PEG ligation reaction mix[edit]

Components Volume (ul) 9x rxn mix
dA-tailed reaction 20.00 0.00
10X Tango buffer 0.50 4.50
HC T4 DNA ligase (30units/ul) 1.00 9.00
10mM ATP 1.25 11.25
H2O 1.25 11.25
Total 24.00 36.00

+ PEG ligation reaction mix[edit]

Components Volume (ul) 9x rxn mix
dA-tailed reaction 20.00 0.00
10X Tango buffer 0.50 4.50
HC T4 DNA ligase (30units/ul) 1.00 9.00
10mM ATP 1.25 11.25
50% PEG 4000 1.25 11.25
Total 24.00 36.00
- Add 1ul of diluted methylated adapter
- Add 4ul of ligation reaction mix
- Mix by gently vortexing & spin down
- Incubate at 22C for 1h or 16C for 16h

Bisulfite conversion[edit]

  • I performed bisulfite conversion exactly as described on 2014-02-14: [[3]]

PCR amplification[edit]

1st round amplification[edit]

Components Conc unit Final conc. unit Volume (ul) 10 rxn mix
Bis-cvt DNA 30.00 0.00
10X Reaction buffer 10 X 1 X 5.00 50.00
dNTP mix 10 mM 0.2 mM 1.00 10.00
PCR_F 10 uM 0.1 uM 0.50 5.00
PCR_N2.Ind3 10 uM 0.1 uM 0.50 5.00
PfuTurbo Cx 2.5 Unit/ul 1 unit 0.40 4.00
50X SYBG 50 X 1 X 1.00 10.00
H2O 11.60 116.00
- Aliquot 20ul, add 30ul of DNA template
- 95C for 2min --> [95C for 20sec -> 65C for 30sec -> 72C for 1min] X 25 cycles --> 72C for 2min
- I took 4ul of the first round PCR amplicons to verify in the gel
  • I prepared the same master mix in both 1st and 2nd round PCR for reactions ligated at 16C for 16h.
- Perform two rounds AMPure bead purification

AMPure bead purification[edit]

- Add 46ul AMPure bead. Mix by pipetting 10x
- Sit for 8min
- Transfer to sit on magnet for 3min
- Wash twice with 150ul freshly prepared 80% EtOH
- Dry the bead for 3-5min
- Resuspend with 40 H2O
- Add 40ul AMPure bead. Mix by pipetting 10x
- Sit for 8min
- Transfer to sit on magnet for 3min
- Wash twice with 150ul freshly prepared 80% EtOH
- Dry the bead for 3-5min
- Resuspend with 30ul H2O

2nd round amplification[edit]

Components Conc unit Final conc. unit Volume (ul) 10 rxn mix
Purified 1st round DNA 30.00 0.00
5X Phusion HF buffer 5 X 1 X 10.00 100.00
dNTP mix 10 mM 0.2 mM 1.00 10.00
PCR_F 10 uM 0.2 uM 1.00 10.00
PCR_N2.Ind2 10 uM 0.2 uM 1.00 10.00
50X SYBR 50 X 1 X 1.00 10.00
Phusion HF 2 unit/ul 0.50 5.00
H2O 5.50 55.00
- Aliquot 20ul, add 30ul of DNA template
- 98C for 2min --> [98C for 10sec -> 65C for 30sec -> 72C for 1min] X 22 cycles --> 72C for 2min
- I monitored qPCR and have taken 4ul of PCR product at different cycle number as shown in the results.

RESULTS[edit]

1st round amplification[edit]

- Load 4ul of 1st round amplicon in 6% TBE gel

File:ZhangLab 2 2014-02-18 12hr 34min 1stround Exp3 22C-1hr.jpg File:ZhangLab 2 2014-02-18 18hr 30min 1stround Exp3 16C-16h.jpg

2nd round amplification[edit]

  • I monitored qPCR and sampling 4ul of PCR product from different cycle number to see the optimal cycle numbers of each nuclei number. 0, 10, 100, and 1000 should reach optimum condition at different cycle number.
Ligation at 22C for 1h result[edit]

File:ZhangLab 2 2014-02-18 16hr 23min 2ndround Exp3 17cycles 22C-1h e.jpg File:ZhangLab 2 2014-02-18 16hr 23min 2ndround Exp3 22cycles 22C-1h e.jpg

Ligation at 16C for 16h result[edit]

File:2ndround Exp3 10cycles 16C-16h e.jpg File:2ndround Exp3 12cycles 16C-16h e.jpg

File:2ndround Exp3 14cycles 16C-16h e.jpg File:2ndround Exp3 16cycles 16C-16h e.jpg

DISCUSSION WILL BE ADDED[edit]

  • Continued on: [[4]]