Noi/NOTES/2014-4-23
scRRBS experiment: Apr#2[edit]
Link to calendar: [[1]]
- Since the result from Experiment Apr#1 looked so promising, I continued to repeat experiment to confirm that I can get consistent result.
- I spiked in unmethylated lambda DNA incase the experiment works well and I can continue to sequencing. I then can monitor bisulfite conversion rate. I can also observe the gel intensity comparing between with and without lambda DNA
NOTE[edit]
- I used the same plate of flow-sorted nuclei as experiment Apr#1 sorted on 2014-04-10 (the 2nd strip of devisable 96-well plate, column #4-6).
- I included 60fg of unmethylated lambda DNA
Experimental Procedures[edit]
Plate layout[edit]
' 4 5 6 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
1) Cell lysis[edit]
Prep
- Thaw nuclei from -80C & spin down at 2,000rpm for 5min (96-well plate rotor, 5min)
- Mix 30ul of QIAGEN Protease with 1.5ul of 1.2pg/ul lambda DNA
- - Add 1.05ul of protease/lambda DNA 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
- - I tried to avoid pipetting up and down to mix the reaction to prevent nuclei/DNA lost.
- - I notice solution was mixed well 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[edit]
- 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
- - Heat inactivate at 65C for 20min <NEW>
- - 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[edit]
- 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
- Prepare new tube of ACG mix
dNTPs | Stock (mM) | Dilute to (mM) | Volume (ul) |
dATP | 100 | 20.00 | 100.00 |
dCTP | 100 | 2.00 | 10.00 |
dGTP | 100 | 2.00 | 10.00 |
H2O (ul) | 380.00 | ||
Total (ul) | 500.00 |
- 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
- - 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[edit]
- 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
Index list
- I used diluted methylated adaptors prepared on 2014-04-21
- Since there was something wrong with Ind_12 in tube_6, I will remove all solution and add 1:20 diluted Ind_5
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_5 | Ind_2 | Ind_4 |
Prep
- Mix 1ul of barcoded methylated adaptors Ind_5 with 20ul H2O (on ice box) and add to tube_6
- 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 12h (no heat lid)
- - 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 # A4 Ind_2 #1 A5 Ind_2 #9 A6 Ind_2 #17 B4 Ind_4 #2 B5 Ind_4 #10 B6 Ind_4 #18 C4 Ind_5 #3 C5 Ind_5 #11 C6 Ind_5 #19 D4 Ind_6 #4 D5 Ind_6 #12 D6 Ind_6 #20 E4 Ind_7 #5 E5 Ind_7 #13 E6 Ind_7 #21 F4 Ind_5 #6 F5 Ind_5 #14 F6 Ind_5 #22 G4 Ind_2 #7 G5 Ind_2 #15 G6 Ind_2 #23 H4 Ind_4 #8 H5 Ind_4 #16 H6 Ind_4 #24
2014-04-24
5) Bisulfite conversion[edit]
- I performed bisulfite conversion using the same procedure following manufacturer's instruction and elute with 31ul elution buffer.
Prep
- Prepare 3 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 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. 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
6) PCR amplification[edit]
Prep
- Prepare 10uM of TruS_F and TruS_R mix volume 500ul by mixing 50ul each of 100uM TruS_F and 100uM TruS_R with 400ul H2O
1st round PCR[edit]
Prep
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/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 11.30 293.80
- - 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 AMPure beads 2X (1:1 ratio)
AMPure bead purification[edit]
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 50ul 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[edit]
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/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 0.50 13.00 H2O 4.20 109.20
- - 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 plan to monitor 100nu and 20nu to stop them earlier since the amplification should be too saturated at 22cycles of the 2nd round PCR.
Results[edit]
- - For 2nd round PCR, I stopped 100nu and 20nu at 19 cycles and the rest at 22 cycles
- - Loaded 4ul of unpurified 2nd round amplicons in 6% TBE gel. This time I load 1nu on the same gel with 0nu for intensity comparison
Ligation condition:
- - 1ul of 1:20 diluted Illumina barcoded adaptors
- - 16C for 30min -> 4C for 12h
File:ZhangLab 2 2014-04-25 00hr 20min 2ndPCR gel1.jpg File:ZhangLab 2 2014-04-25 00hr 20min 2ndPCR gel2.jpg
File:ZhangLab 2 2014-04-25 00hr 20min 2ndPCR gel3.jpg
- ** : It's likely that adaptors were not added added to this well since there was no adaptor dimer band ~125bp.
Summary of number of positive samples[edit]
- With lambda DNA spike in
- - 1nu: #6 (Ind_5)
- - 100nu: #9 (Ind_2 ), #10 (Ind_4)
- - 20nu: #11 (In_5), one sample faile --> no adaptor in this well (from gel image)
- - 10nu: #13 (Ind_7), #14 (Ind_5)
- - I do bead purified on these samples (44ul, 1:1 ratio)
- - Elute with 50ul EB buffer
- - Load 5ul in 6% TBE gel for PAGE quantification
- #6, #9, #10, #11, #13, #14
Sample # Index # of nuclei Sample ID #6 5 1 Apr2_#6_1_5 #9 2 100 Apr2_#9_100_2 #10 4 100 Apr2_#10_100_4 #11 5 20 Apr2_#11_20_5 #13 7 10 Apr2_#13_10_7 #14 5 10 Apr2_#14_10_5