Noi/NOTES/2014-4-21

From ZhangLabWiki
Revision as of 00:27, 24 April 2014 by >Noi (→‎Results)
Jump to navigation Jump to search

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

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



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:ZhangLab 2 2014-04-23 13hr 46min Apr

File:ZhangLab 2 2014-04-23 13hr 46min Apr

File:ZhangLab 2 2014-04-23 13hr 46min Apr

Discussions