Noi/NOTES/2014-12-17
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scRBS experiment of single neuronal nucleus
- 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