Noi/NOTES/2014-4-21
scRRBS experiment Apr#1
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- I stopped doing scRRBS experiment for a while since I never get it works for single nuclei, and I think I should use the same barcoded methylated adaptors as used by Dr. Tang's 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 differently from them. To be able to repeat the protocol and get it works 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 reason that I never get success in single-nuclei experiment as I alway stopped reaction during the 2nd round PCR earlier before reaching 22 cycles (between 12-15 based on qPCR curve)
- They heat-inactivate after MspI digestion, end-repair/dA-tailing, and methylated adaptors 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.
- For PCR, thy used 200uM of dNTP and 300nM of primer-pair
- I share all of these info with Yun Liu, the postdoctoral fellow in Dr. Feinberg's group, since he wants to try this protocol too.
NOTE
- I used flow-sorted nuclei on 2014-04-10 for this experiment.
- I still skipped 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
- I increased primer concentration in both 1st round and 2nd round PCR to 0.3mM or 300uM
Experimental Procedures
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.
- Some steps were not the same as Dr. Tang'g group suggestion since I got his email after I started experiment.
1) Cell lysis
Prep
- Thaw nuclei from -80C & spin down at 2000rpm for 5min (96-well plate rotor, 5min)
- - Add 1ul of protease
- - 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 notice solution was mixed and no foaming generated
- - 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
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) 26x rxn mix Lysed nuclei 5.00 0.00 10X Tango buffer 2.00 52.00 MspI 0.90 23.40 H2O 10.10 262.60 Total 18.00 338.00
- Aliquot 42ul of MspI enzyme mix to each tube in 8-tube strip
- - 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
- - Set program to hold at 15C
- - 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
- Aliquot 5ul of dA:dC:dG mix (20mM:2mM:2mM) to each tube of 8-tube strip
- Aliquot 3.5ul of Klenow fragment exo- to each tube of 8-tube strip
- - Add 1ul of dA:dC:dG solution mix to each well with multichannel pipette
- - Add 1ul of Klenow fragment exo- to each well with multichannel pipette
- - 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
- - I prefer to add reagents with multichannel pipette since I want to make sure no samples were missing during adding reagents and save a lot of my time. Indeed, less pipetting can avoid contamination.
- - 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 1:20 diluted Illumina indexed methylated adaptor in total reaction 25ul at 16C for 30min and 4C for at least 8h (I did 14.5h --> next time will fix incubation time at 4C for consistency).
Index list
- I have only 6 indexes, I put Ind_2 and Ind_4 twice in 8-tube strip
Tube_1 | Tube_2 | Tube_3 | Tube_4 | Tube_5 | Tube_6 | Tube_7 | Tube_8 |
Ind_2 | Ind_4 | Ind_5 | Ind_6 | Ind_7 | Ind_12 | Ind_2 | Ind_4 |
Prep
- Mix 1ul of indexed methylated adaptors with 20ul H2O (on ice box)
- Prepare ligation reaction mix
Components | Volume (ul) | 28x rxn mix |
dA-tailed reaction | 20.00 | 0.00 |
10X Tango buffer | 0.50 | 14.00 |
HC T4 DNA ligase (30units/ul) | 1.00 | 28.00 |
10mM ATP | 1.25 | 35.00 |
H2O | 1.25 | 35.00 |
Total | 24.00 | 112.00 |
- Aliquot 14ul of Ligation mix to each tube in 8-tube strip
- - Add 1ul of diluted methylated adapter
- - Add 4ul of ligation reaction mix
- - 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 for 30min -> 4C for 14.5 (at least 8h)
- - Heat inactivate at 65C for 20min
- - Spin down the plate at 2,000rpm for 1min before continuing to next step
Well ID Index Sample # Well ID Index Sample # Well ID Index Sample # A1 Ind_2 #1 A2 Ind_2 #9 A3 Ind_2 #17 B1 Ind_4 #2 B2 Ind_4 #10 B3 Ind_4 #18 C1 Ind_5 #3 C2 Ind_5 #11 C3 Ind_5 #19 D1 Ind_6 #4 D2 Ind_6 #12 D3 Ind_6 #20 E1 Ind_7 #5 E2 Ind_7 #13 E3 Ind_7 #21 F1 Ind_12 #6 F2 Ind_12 #14 F3 Ind_12 #22 G1 Ind_2 #7 G2 Ind_2 #15 G3 Ind_2 #23 H1 Ind_4 #8 H2 Ind_4 #16 H3 Ind_4 #24
2014-04-22
5) Bisulfite conversion
- I performed bisulfite conversion using the same procedure following manufacturer's instruction and elute with 31ul elution buffer.
Prep
- Prepare 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
- - 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 24 rxn, I mixed 14.7mL of Binding Buffer with 24.5 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) 31ul Elution Buffer. This should have ~30ul DNA left for PCR
6) PCR amplification
- Primer info.
- - TruS_F: 5'- AATGATACGGCGACCACCGAGATC -3' (Tm = 67.1 C)
- - TruS_R: 5'- CAAGCAGAAGACGGCATACGAGAT -3'(Tm = 65.5 C)
- - I designed PCR primers a little longer (~3bp) than the ones used in Boyle P et al, 2012
1st round PCR
Components Conc unit Final conc. unit Volume (ul) 26 rxn mix Bis-cvt DNA 30.00 0.00 10X Reaction buffer 10 X 1 X 5.00 130.00 dNTP mix 10 mM 0.2 mM 1.00 26.00 TruS_F 10 uM 0.3 uM 1.50 39.00 TruS_R 10 uM 0.3 uM 1.50 39.00 PfuTurbo Cx 2.5 Unit/ul 1 unit 0.40 10.40 50X SYBG 50 X 0.8 X 0.80 20.80 H2O 9.80 254.80
- - Aliquot 20ul, add 30ul of bisulfite-treat adaptor-ligated DNA
- - 95C for 2min --> [95C for 20sec -> 60C for 30sec -> 72C for 1min] X 25 cycles --> 72C for 2min
- - 95C for 2min --> [95C for 20sec -> 60C for 30sec -> 72C for 1min] X 25 cycles --> 72C for 2min
- - Purified the 1st round amplicons with AMPurebeads 2X (1:1 ratio)
AMPure bead purification
Prep
- Freshly prepare 20mL of 75% EtOH by mixing 15mL of 100% EtOH with 5mL of H2O
- Add ~2.5mL of resuspened AMPure bead in 30mL reservoir
- - Add 40ul AMPure bead. Mix by pipetting 10x
- - Sit for 8min
- - Transfer to sit on magnet for 5min
- - Wash twice with 160ul freshly prepared 80% EtOH
- - Dry the bead for 3-5min
- - Resuspend with 50 H2O
- - Add 50ul of fresh AMPure bead. Mix by pipetting 10x
- - Sit for 5min
- - Transfer to sit on magnet for 5min
- - Wash twice with 160ul freshly prepared 80% EtOH
- - Dry the bead for 3-5min (make sure that the beads are completely dried out to avoid EtOH inhibiting PCR)
- - Resuspend the bead with 40ul H2O
- - Transfer purified 1st round amplicons to 8-tube strip with cap
- - Sit the strip tube on magnet before adding to the 2nd round PCR to avoid bead contamination in PCR
- - 32ul of bead purified 1st round amplicons will be added to the 2nd round PCR (32/40 -> 40%)
- - I saved the rest of bead purified 1st round amplicons
2nd round PCR
Components Conc unit Final conc. unit Volume (ul) 26 rxn mix Purified 1st round DNA 32.00 0.00 5X Phusion HF buffer 5 X 1 X 10.00 260.00 dNTP mix 10 mM 0.2 mM 1.00 26.00 TruS_F 10 uM 0.3 uM 1.50 39.00 TruS_R 10 uM 0.3 uM 1.50 39.00 50X SYBR 50 X 0.8 X 0.80 20.80 Phusion HF 2 unit/ul 1 unit 0.50 13.00 H2O 2.70 70.20
- - Aliquot 18ul, add 32ul of bead purified 1st round amplicons
- - 98C for 2min --> [98C for 10sec -> 60C for 30sec -> 72C for 1min] X 22 cycles --> 72C for 2min
- - 98C for 2min --> [98C for 10sec -> 60C for 30sec -> 72C for 1min] X 22 cycles --> 72C for 2min
- - I did qPCR but let the reaction run to 22 cycles for all samples.
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:Fgh e.png
- Since I can not put all samples in the same gel, I stained the gels for 4min then replaced staining solution with 0.5X TBE buffer. I also used the same set up for all images