Noi/NOTES/2014-12-17

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scRBS experiment of single neuronal nucleus

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  • This experiment, I will generate RRBS libraries of mouse single nuclei, including control:wild-type; and TKO: Dnmt1; 3a; 3b. Youjin from UCLA will pick single nuclei under microscope for this experiment. The method for cell picking would be different from previous samples I got from Scripp.
  • NOTE that I have performed two experiments, including this experiment and one on 2014-12-20. The first experiment (this experiment) seemed to fail and difficult to interpret the result. However, I would document all details in each experiment for comparison the conditions with the one that we think it worked well.

Cell picking

  • Youjin preferred to have lysis buffer (as bedding buffer) in 0.2mL PCR tube as it's convenient for him to handle one tube at a time and to avoid contamination.
  • He said that the volume of picked nucleus and buffer should be ~0.5ul, so I prepare 3.5ul of 1.43 x Lysis buffer to get a final concentration of 1x after loading 0.5ul of picked nucleus and 1ul of protease.
  • Since cell picking finished pretty late, so I stored picked nuclei in -80C and continued cell lysis on the following day.

1) Cell lysis

Prep
- Thaw nuclei from -80C & spin down at 2,000rpm for 5min. I put PCR tube on PCR rack. (96-well plate rotor, 5min)
- Mix QIAGEN Protease with lambda DNA. I prepared more than enough of protease and unmethy lambda DNA mix.

Components 1X rxn 70X rxn
Cell in 1.43X lysis buffer 4.00 0.00
1.2pg Lambda DNA 0.05 3.50
Protease 1.00 70.00
Total 5.05
  • Final amount of unmet lambda DNA in each tube is 60fg.

Sample list

  • WT = wild type
  • T= TKO
  • Number after WT or T like W10, T100 is numb rod nuclei the rest are W or T with single nucleus and running order number.
WT_1 WT_2 WT_3 WT_4 WT_5 WT_6 WT_7 WT_8
WT_9 WT_10 WT_11 WT_12 T_1 T_2 T_3 T_4
T_5 T_6 WT10_1 WT10_2 WT100 0nu NTC
T_7 T_8 T_9 T_10 T_11 T_12 T_10 T100
NTC 0nu
  • I handled sample WT-1 --> NTC (23 samples)
  • Youjin handled sample T7 --> 0nu (10 samples)
  • I prepared the same master mix then aliquot for Youjin for every step.
  • Note that Youjin did not pick 0 nuclei from the nucleus suspension which is ideal negative control to make sure that the signals do not come from buffer or other sources. We therefore use lysis buffer that I aliquot as bedding buffer as 0nu control.
  • For NTC, I aliquot lysis buffer from original tube of lysis buffer and add H2O instead of pick nuclei.
- 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 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
  • Note that before cell lysis and after cell lysis before continue to MspI digestion, I need to spin the tube containing single nucleus at high speed and long time to make sure that I do not lose nucleus before cell lysis.


2) 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) 34x rxn mix
Lysed nuclei 5.00 0.00
10X Tango buffer 2.00 68.00
MspI 0.90 30.60
H2O 10.10 343.40
Total 18.00
- 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
- Heat inactivate at 65C for 20min
- Set program to hold at 4C
- Spin down the plate at 2,000rpm for 1min before continuing to next step


3) Gap-filling/dA-tailing