Noi/NOTES/2014-12-17
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scRBS experiment of single mouse 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
3) 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
- - Mix 35 dA:dC:dG mix (20mM:2mM:2mM) with 35ul of Klenow fragment exo- (just before adding to MspI-digested DNA)
- - Add 2ul of dA:dC:dG/Klenow fragment exo- mix to each tube. Do not need to touch the reaction. Just touch the tip of pipette tip close enough to the reaction and deliver reaction mix.
- - 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 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
4) Methylated adaptor ligation
- Ligate A-tailed DNA with 1ul of 1:50 diluted Illumina indexed methylated adaptor in total reaction 25ul at 16C for 30min and 4C for at least 16h.
- We have modified to incubate at 16C O/N
- For this round of experiment, Youjin preferred to use STD Illumina adaptor as he wanted to include all samples in the same lane for sequencing in case that the experiment works well.
Components | Volume (ul) | 36x rxn mix |
dA-tailed reaction | 20.00 | 0.00 |
10X Tango buffer | 0.50 | 18.00 |
HC T4 DNA ligase (30units/ul) | 1.00 | 36.00 |
10mM ATP | 1.25 | 45.00 |
15uM Univ adaptor | 0.02 | 0.72 |
H2O | 2.23 | 80.28 |
Total | 25.00 |
- - Add 5ul of ligation reaction mix with 12nM of STD Illumina adaptor
- - 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 O/N (~18h, no heat lid)
- - Heat inactivate at 65C for 20min. This time I skipped this step
- - Spin down the plate at 2,000rpm for 1min before continuing to next step
5) Bisulfite conversion
- I performed bisulfite conversion using the same procedure following manufacturer's instruction and elute with 32ul elution buffer.
Prep
- Prepare 4 tubes of 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 with multi-channel pipette
- - 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 33.5 rxn, I mixed 20.10mL of Binding Buffer with 33.5 ul of 10ng/ul tRNA
- - 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 DNA to the column as much as possible
- - 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) 32ul Elution Buffer. Incubate column with Elution Buffer at room temp for 2min.
- - Spin down at 14,000rmp for 1min. This should have ~30ul DNA left for PCR