Noi/NOTES/2014-4-21

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scRRBS experiment Apr#1

  • I stopped doing scRRBS experiment for a while since I never get it work for single nuclei, and I think I should use the same barcoded adaptors as used by Dr. Tang group.
  • I found an old TrueSeq DNA Sample Prep kit in (Set A, part # 15013178) -20C. I then wanted to try these barcoded adaptors for ssRRBS while waiting for TrueSeq kit requested by Dr. Zhang. In this kit, I found only 6 indexed adaptors, including Ind2, Ind 4, Ind 5, Ind 6, Ind7 and Ind 12.
  • I have contacted with Dr. Tang's group for more information of scRRBS protocol in very details, and found there are many things I did different from them. To be able to repeat the protocol and get it work for single cell, I followed everything noted by them.

More information from Dr. Tang's group

  • They used 80% of bead purified (two-round purification) 1st round amplicons as the template for the 2nd round PCR.
  • In both the 1st round and the 2nd round PCR, they did not do qPCR to monitor amplicons. They said that that the bisulfite-treated DNA was extremely low, so they did not monitor it.
- I think this might be the key that I never get success in single-nuclei experiment as I alway stopped reaction during the 2nd round PCR earlier before reaching 22 cycles (like 12 or 15 based on qPCR curve)
  • They heat-inactivate after MspI digestion, end-repair/dA-tailing, and ligation
- MspI (not specify)
- End-repair/dA-tailing (I did 75C, 20min
- Ligation (65C, 20min) --> from Gu et al, 2011 (Nature Protocol). They suggest not to heat the lid since I could potentially destroy T4 DNA ligase.
  • They incubate ligation reaction at 16C, 30min --> 4C (at least 8hr). I previously incubate at 16C for at least 12h.
  • I share all of information these info with Yun Liu, the postdoc fellow in Dr. Feinberg group, since he wants to try this protocol too.

  • I used flow-sorted nuclei on 2014-04-10 for this experiment.
  • I still skip unmethylated lambda DNA since I want to avoid the background in trial experiment. Once the protocol is settle, I will include it to see bisulfite conversion rate

Experimental Procedures

Plate layout

' 1 2 3
A 1 100 0
B 1 100 0
C 1 20 0
D 1 20 0
E 1 10 0
F 1 10 0
G 1 1 0
H 1 1 0
  • I include many of 0 nuclei since I want to see the consistency in all of them.
  • Note: Some steps were not the same as Dr. Tang'g group suggestion since I got his emailed after I started experiment.

Cell lysis

- Thaw nuclei from -80C & spin down at 2000rpm for 5min (96-well plate rotor, 5min)
- Add 1ul of protease
- Spin down the plate at 2000rpm for m2min
- Mix by pulse gently vortexting for 10x.
- Spin down the plate at 2000rpm for 3min
- I tried to avoid pipetting up and down to mix the reaction to prevent nuclei/DNA lost.
- I notice solution was mixed and no foaming generated
- I spin down the plate before and after mixing quite long to make sure that all reagents were collected to the bottom of the well
- Incubate at 50C for 3hr
- Heat inactivate at 75C for 30min
- Set program to hold at 15C
- Spin down the plate at 2000rpm for 1min before continuing to next step

DNA fragmentation with MspI

  • Incubated released naked DNA with 9units of MspI in 18ul reaction at 37C for 3hr.
Components Volume (ul) 26x rxn mix
Lysed nuclei 5.00 0.00
10X Tango buffer 2.00 52.00
MspI 0.90 23.40
H2O 10.10 262.60
Total 18.00 338.00
- Aliquot 42ul of MspI enzyme mix to each tube in 8-tube strip
- Add 13ul to each well with multi-channel pipetter
- Spin down the plate at 2000rpm for m2min
- Mix by pulse gently vortexting for 10x.
- Spin down the plate at 2000rpm for 3min
- Incubate at 37C for 3hr
- Set program to hold at 15C
- Spin down the plate at 2000rpm for 1min before continuing to next step

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)
- Aliquot 5ul of dA:dC:dG mix (20mM:2mM:2mM) to each tube of 8-tube strip
- Aliquot 3.5ul of Klenow fragment exo- to each tube of 8-tube strip
- Add 1ul of dA:dC:dG solution mix to each well with multichannel pipette
- Add 1ul of Klenow fragment exo- to each well with multichannel pipette
- Add 13ul to each well with multi-channel pipette
- Spin down the plate at 2000rpm for m2min
- Mix by pulse gently vortexting for 10x.
- Spin down the plate at 2000rpm for 3min
- I prefer to add reagents with multichannel pipette since I want to make sure no samples were missing during adding reagents and save a lot of my time. Indeed, less pipette work can avoid contamination.
- Incubate at 30C for 20min (for gap-filling)
- Incubate at 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 2000rpm for 1min before continuing to next step

Methylated adaptor ligation

  • Ligate A-tailed DNA with 1ul of 1:20 diluted Illumina indexed methylated adaptor in total reaction 25ul at 16C for 30min and 4C for at least 8h (I did 14.5h --> next time will fix incubation time at 4C for consistency).

Index list
- I have only 6 indexes, I put Ind_2 and Ind_4 twice in 8-tube strip

Tube_1 Tube_2 Tube_3 Tube_4 Tube_5 Tube_6 Tube_7 Tube_8
Ind_2 Ind_4 Ind_5 Ind_6 Ind_7 Ind_12 Ind_2 Ind_4

Prep
- Mix 1ul of indexed methylated adaptors with 20ul H2O (on ice box)
- Prepare ligation reaction mix

Components Volume (ul) 28x rxn mix
dA-tailed reaction 20.00 0.00
10X Tango buffer 0.50 14.00
HC T4 DNA ligase (30units/ul) 1.00 28.00
10mM ATP 1.25 35.00
H2O 1.25 35.00
Total 24.00 112.00

- Aliquot 14ul of Ligation mix to each tube in 8-tube strip

- Add 1ul of diluted methylated adapter
- Add 4ul of ligation reaction mix
- Spin down the plate at 2000rpm for 2min
- Mix by pulse gently vortexting for 10x.
- Spin down the plate at 2000rpm for 3min
- Incubate at 16C for 30min -> 14C for 14.5 (at least 8h)
- Heat inactivate at 65C for 20min
- Spin down the plate at 2000rpm for 1min before continuing to next step
A1 Ind_2 A2 Ind_2 A3 Ind_2
B1 Ind_4 B2 Ind_4 B3 Ind_4
C1 Ind_5 C2 Ind_5 C3 Ind_5
D1 Ind_6 D2 Ind_6 D3 Ind_6
E1 Ind_7 E2 Ind_7 E3 Ind_7
F1 Ind_12 F2 Ind_12 F3 Ind_12
G1 Ind_2 G2 Ind_2 G3 Ind_2
H1 Ind_4 H2 Ind_4 H3 Ind_4

Bisulfite conversion

  • I performed bisulfite conversion using the same procedure following manufacturer's instruction and elute with 31ul elution buffer.
- Resuspend CT conversion reagent as described in instruction
- Note: Add 850ul H2O instead of 900ul to adjust final concentration of CT conversion reagent the same as 20ul sample input during bisulfite conversion
- Mix 25ul of DNA sample with 125ul of CT Conversion Reagent (no sample transfer to the new tube)
- 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
- Bind DNA to column by mixing with 600ul Binding Buffer and 1ul of 10ng/ul tRNA as carrier. Spin down 14,000rpm for 30sec
- For 24 rxn, I mix 14.7mL of binding buffer with 24.5 ul of 10ng/ul tRNA
- 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 minsec
- Elute converted DNA with warm (~60C) 31ul Elution Buffer. This should have ~30ul DNA left for PCR

Results

- Purified 40ul of 2nd round PCR with 1:1 ratio
- Eluted with 40ul EB buffer
- Loaded 4ul of bead purified PCR products and also load 4ul some of unpurified PCR products

File:Abc.png

File:Cde.png

File:Fgh e.png

Discussions