Noi/NOTES/2014-12-20
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scRBS experiment of single mouse neuronal nucleus (Exp #2)
- This experiment continued from the the first of experiment (Exp#1) for UCLA (Dr. Fan's group)
- I had some discussion in previous experiment. For this experiment, I will MONITOR PCR in the 1st PCR carefully to prevent over-amplification of adaptor dimers.
- In addition, I will include more control either positive control and negative control in every step to make sure that where the signal comes from. I will also include and exclude unmeth lambda DNA as well.
Sample list in each step in brief
Cell lysis
- - WT_1 to WT_16 (WT 1 nu, performed by Youjin). I re-run the number of samples to make it easy to handle and track.
- For my part I will include all controls and also in the following steps
- - T_1 to T_16 (TKO 1 nu, performed by Noi).
- - T10, T100 and T0 (TKO 10nu, 100nu and 0nu, 1 each, performed by Noi)
- - 10pg of CTT with unmeth lambda DNA (duplicates). CTT is gDNA from frozen colon tumor tissue purchased from Biochain.
- - NTC with unmeth lambda DNA (duplicate),
- All samples in this step will have 60fg of unmeth lambda DNA spike in. This allows for bisulfite conversion efficiency measurement.
- - I did serial dilution of CTT gDNA from 2ng/ul to 100pg/ul and 5pg/ul
- - Mix 10ul of 2ng/ul CTT gDNA with 190ul H2O
- - Mix 10ul of 100pg/ul CTT gDNA with 190ul H2O
DNA fragmentation with MspI
- - 10pg of CTT without unmeth lambda DNA (duplicates).
- - MspI NTC without unmeth lambda DNA (duplicates).
Methylated adaptor ligation
- - Ligation control (duplicates).
- No NTC during Gap-filling/dA-tailing
Experimental procedure
- I used mouse neuronal nuclei picked on 2014-12-17 and stored at -80C
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 100X rxn Cell in 1.43X lysis buffer 4.00 0.00 1.2pg Lambda DNA 0.05 5.00 Protease 1.00 100.00 Total 5.05
- Final amount of unmet lambda DNA in each tube is 60fg.
- - Add 1.05 ul of protease and unmeth lambda DNA mix to each tube. No need to pipette!
- - Spin down the tube on PCR rack 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 tube
- - 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 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.
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) 42x rxn mix Lysed nuclei 5.00 0.00 10X Tango buffer 2.00 84.00 MspI 0.90 37.80 H2O 10.10 424.20 Total 18.00
- - Set up positive control of 10pg CTT gDNA (no lambda DNA) and MspI NTC (no lambda DNA) in duplicates
- - 10pg CTT: Add 2ul of 5pg/ul CTT gDNA and 3ul H2O
- - MspI NTC, Add 5ul H2O
- - Aliqout 13ul of MspI reaction mix to each tube
- - 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) End-repair or 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 50 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 1:25 diluted Illumina indexed methylated adaptor (TruSeq adaptor) in total reaction 25ul at 16C for 30min and 4C for at least 16h.
- Final conc. of adaptor in 25ul ligation reaction is ~23nM
- In Dr. Tang's paper, he used 1ul of 1:20 diluted TruSeq adaptor (final conc. ~28.5nM
- In this experiment, I made fresh batch of 1:25 diluted TruSeq adaptor in 0.2mL 8-tube strip by mixing 2ul of 15uM of TruSeq adaptor with 50ul H2O
Components Volume (ul) 50x rxn mix dA-tailed reaction 20.00 0.00 10X Tango buffer 0.50 25.00 HC T4 DNA ligase (30units/ul) 1.00 50.00 10mM ATP 1.25 62.50 H2O 1.25 62.50 Total 24.00
- - Add 1ul of 1:25 diluted TruSeq adaptor to each well with multichannel pipette
- - Add 4ul of ligation reaction mix to each tube
- - 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
- Note: The way I assigned TruSeq index was based on Youjin suggestion. The reason was that we had limited number of indexed barcode (24), and Youjin wanted to combined both WT and TKO for sequencing in the same lane. This is why I used duplicated indexes for sample in the same group (WT or TKO) and have at least 12 different indexes between the two groups (Index 1-12 for WT and Index 13-27 for TKO, no index #17, 24, and 26.
6) Bisulfite conversion
- I performed bisulfite conversion using the same procedure following manufacturer's instruction and elute with 32ul elution buffer.
Prep