Noi/NOTES/2014-4-21: Difference between revisions
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* I include many of 0 nuclei since I want to see the consistency in all of them. | * I include many of 0 nuclei since I want to see the consistency in all of them. | ||
=== Cell lysis === | |||
:- Thaw nuclei from -80C & spin down at 2000rpm (96-well plate rotor, 5min) | |||
:- Mix protease and 1.2 pg/ul unmethylated lambda DNA 1ul:0.05 ratio (30ul + 1.5ul). This will be used 1.05ul for nuclei lysis. | |||
:- Add 1.05ul of protease/unmethylated lambda DNA mix | |||
:- ''Incubate at 50C for 3hr''<br> | |||
:- ''Heat inactivate at 75C for 30min'' | |||
<br> |
Revision as of 23:38, 23 April 2014
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 2014-03-18 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
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.
Cell lysis
- - Thaw nuclei from -80C & spin down at 2000rpm (96-well plate rotor, 5min)
- - Mix protease and 1.2 pg/ul unmethylated lambda DNA 1ul:0.05 ratio (30ul + 1.5ul). This will be used 1.05ul for nuclei lysis.
- - Add 1.05ul of protease/unmethylated lambda DNA mix
- - Incubate at 50C for 3hr
- - Heat inactivate at 75C for 30min