Noi/NOTES/2014-4-21: Difference between revisions

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:- Mix by gentle pulse-vortexting for 10x
:- Mix by gentle pulse-vortexting for 10x
:- Spin down the plate at 2,000rpm for 3min
:- Spin down the plate at 2,000rpm for 3min
:- ''Incubate at 16C for 30min -> 14C for 14.5 (at least 8h)''
:- ''Incubate at 16C for 30min -> 4C for 14.5 (at least 8h)''
:- ''Heat inactivate at 65C for 20min''
:- ''Heat inactivate at 65C for 20min''
:- Spin down the plate at 2,000rpm for 1min before continuing to next step  
:- Spin down the plate at 2,000rpm for 1min before continuing to next step  

Revision as of 06:02, 24 April 2014

scRRBS experiment Apr#1

Link to calendar: [[1]]

  • I stopped doing scRRBS experiment for a while since I never get it works for single nuclei, and I think I should use the same barcoded methylated adaptors as used by Dr. Tang's 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 differently from them. To be able to repeat the protocol and get it works 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 reason that I never get success in single-nuclei experiment as I alway stopped reaction during the 2nd round PCR earlier before reaching 22 cycles (between 12-15 based on qPCR curve)
  • They heat-inactivate after MspI digestion, end-repair/dA-tailing, and methylated adaptors 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.
  • For PCR, thy used 200uM of dNTP and 300nM of primer-pair
  • I share all of these info with Yun Liu, the postdoctoral fellow in Dr. Feinberg's group, since he wants to try this protocol too.

NOTE

  • I used flow-sorted nuclei on 2014-04-10 for this experiment.
  • I still skipped 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.
  • Some steps were not the same as Dr. Tang'g group suggestion since I got his emailed after I started experiment.

Cell lysis

Prep
- Thaw nuclei from -80C & spin down at 2000rpm for 5min (96-well plate rotor, 5min)

- Add 1ul of protease
- 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
- 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 2,000rpm 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.

Prep
- Prepare MspI reaction mix

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 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
- Set program to hold at 15C
- Spin down the plate at 2,000rpm 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)

Prep
- 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 2,000rpm for 2min
- Mix by gentle pulse-vortexting for 10x
- Spin down the plate at 2,000rpm 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 pipetting can avoid contamination.
- 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


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 gentle pulse-vortexting for 10x
- Spin down the plate at 2,000rpm for 3min
- Incubate at 16C for 30min -> 4C for 14.5 (at least 8h)
- Heat inactivate at 65C for 20min
- Spin down the plate at 2,000rpm 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

2014-04-22

Bisulfite conversion

  • I performed bisulfite conversion using the same procedure following manufacturer's instruction and elute with 31ul elution buffer.

Prep
- Prepare 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
- 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 24 rxn, I mixed 14.7mL of Binding Buffer with 24.5 ul of 10ng/ul tRNA

- Bind DNA to column by mixing with 600ul Binding Buffer and 1ul of 10ng/ul tRNA as carrier.
- 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. I rinse the well with small amount of Binding Buffer to transfer as much as possible of DNA to the column
- 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) 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
- Since I can not put all samples in the same gel, I stained the gels for 4min then replaced staining solution with 0.5X TBE buffer. I also used the same set up for all images

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