Noi/NOTES/2014-4-25
scRRBS experiment: Apr#3[edit]
Link to calendar: [[1]]
- From Apr#2 experiment, the experiment worked very well for 10nu, 20nu, and 100nu.
- However, the successful rate of 1nu was too low. I only got strong signal in 1 out of 10 samples. I am not sure this is about the sorting issue or losing nuclei during cell lysis (by mixing).
- I want to test the condition with more diluted methylated adaptors at 1:50 and 1:100 since I think that using 1:20, the amplification is too saturated at adaptor bands. The yield of 10nu and 20nu will tell the efficiency of ligation by reducing adaptors concentration.
NOTE[edit]
- I used the same plate of flow-sorted nuclei as experiment Apr#1 and Apr#2 sorted on 2014-04-10 (the 3rd and 4th strips of devisable 96-well plate, column #7-9 and 10-12, respectively).
- I included 60fg of unmethylated lambda DNA.
- I included 2x 10nu and 2x 20nu as positive control, 2x 0nu as NTC and 10x 1nu.
Experimental Procedures[edit]
Plate layout[edit]
' 7/10 8/11 9/12 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
- - Column #7-9: 1:50 diluted adaptors
- - Column #10-12: 1:100 diluted adaptors
- There are 16 samples for each condition. Total 32 samples for this experiment.
1) Cell lysis[edit]
Prep
- Thaw nuclei from -80C & spin down at 2,000rpm for 5min (96-well plate rotor, 5min)
- Mix 45ul of QIAGEN Protease with 2.25ul of 1.2pg/ul lambda DNA
- Aliquot protease/lambda DNA into 8-tube strip
- - Add 1.05ul of protease/lambda DNA mix 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 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
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) 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 18.00 442.00
- Aliquot MspI enzyme mix to each tube in 8-tube strip --> tube_1-2: 28ul, tube_3-6: 54ul, tube_7-8: 82ul
- - 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[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
- Aliquot 50 dA:dC:dG mix (20mM:2mM:2mM) to each tube of 8-tube strip
- Aliquot 20ul 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 16h. (I will fix at this incubation time for every scRRBS experiment).
Index list
- I think I forgot to add ind_12 in the diluted adaptor tube_6, so this experiment I will include it.
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
- Diluted 1:50 of TrueSeq methylated adaptors by mixing 1ul of adaptor with 50ul H2O and mix
- For 1:100 dilution, I mixed 25ul of 1:50 dilution with 25ul H2O
- Prepare ligation reaction mix
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 H2O 1.25 45.00 Total 24.00 144.00
- Aliquot 18ul 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 17h (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 # A7/10 Ind_2 #1 A8/11 XX XX A9/12 XX XX B7/10 Ind_4 #2 B8/11 XX XX B9/12 XX XX C7/10 Ind_5 #3 C8/11 Ind_5 #11 C9/12 Ind_5 #19 D7/10 Ind_6 #4 D8/11 Ind_6 #12 D9/12 Ind_6 #20 E7/10 Ind_7 #5 E8/11 Ind_7 #13 E9/12 XX XX F7/10 Ind_12 #6 F8/11 Ind_12 #14 F9/12 XX XX G7/10 Ind_2 #7 G8/11 Ind_2 #15 G9/12 XX XX H7/10 Ind_4 #8 H8/11 Ind_4 #16 H9/12 XX XX
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 32 rxn, I mixed 19.5mL of Binding Buffer with 32.5 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]
1st round PCR[edit]
Prep
Components Conc unit Final conc. unit Volume (ul) 34 rxn mix Bis-cvt DNA 30.00 0.00 10X Reaction buffer 10 X 1 X 5.00 170.00 dNTP mix 10 mM 0.2 mM 1.00 34.00 TruS_F/R 10 uM 0.3 uM 1.50 51.00 PfuTurbo Cx 2.5 Unit/ul 1 unit 0.40 13.60 50X SYBG 50 X 0.8 X 0.80 27.20 H2O 11.30 384.20
- - 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 26mL of 75% EtOH by mixing 19.5mL of 100% EtOH with 6.5mL of H2O
- Add ~3.5mL 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]
Prep
Components Conc unit Final conc. unit Volume (ul) 34 rxn mix Purified 1st round DNA 32.00 0.00 5X Phusion HF buffer 5 X 1 X 10.00 340.00 dNTP mix 10 mM 0.2 mM 1.00 34.00 TruS_F/R 10 uM 0.3 uM 1.50 51.00 50X SYBR 50 X 0.8 X 0.80 27.20 Phusion HF 2 unit/ul 0.50 17.00 H2O 4.20 142.80
- - 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
Results[edit]
- - For 2nd round PCR, I stopped 20nu at 19 cycles and the rest at 22 cycles
- - Loaded 4ul of unpurified 2nd round amplicons in 6% TBE gel.
Ligation condition:
- - 1ul of 1:50 and 1:100 diluted Illumina barcoded adaptors
- - 16C for 30min -> 4C for 17h
Discussion[edit]
PAGE quantification[edit]
- Purified libraries with 1:1 ratio AMPure beads and eluted with 40ul EB buffer (not all samples).
- Loaded 5ul in 6%TBE gel for quantification
1:50 diluted adaptors
Sample ID | Conce. (ng/ul) | Yield in 35ul |
1:50_#1 | 2.10 | 73.36 |
1:50_#2 | 1.13 | 39.71 |
1:50_#3 | 0.68 | 23.69 |
1:50_#4 | 0.34 | 11.91 |
1:50_#5 | 0.24 | 8.48 |
1:50_#6 | 2.61 | 91.24 |
1:50_#7 | 1.33 | 46.71 |
1:50_#8 | 1.95 | 68.11 |
1:50_#15 | 3.24 | 113.27 |
1:50_#16 | 3.61 | 126.21 |
Sample ID | Conce. (ng/ul) | Yield in 35ul |
1:50_#11 | 7.83 | 273.97 |
1:50_#12 | 7.46 | 260.97 |
1:50_#13 | 7.31 | 255.71 |
1:50_#14 | 9.01 | 315.44 |
1:100_#11 | 5.21 | 182.49 |
1:100_#12 | 2.08 | 72.69 |
1:100_#13 | 5.39 | 188.78 |
1:100_#14 | 6.37 | 223.09 |
1:100 diluted adaptors
Sample ID | Conce. (ng/ul) | Yield in 35ul |
1:100_#1 | 0.12 | 4.26 |
1:100_#2 | 3.15 | 110.32 |
1:100_#3 | 0.45 | 15.89 |
1:100_#4 | 0.77 | 27.07 |
1:100_#5 | 1.19 | 41.80 |
1:100_#6 | 1.46 | 51.03 |
1:100_#7 | 1.00 | 35.16 |
1:100_#8 | 1.74 | 61.01 |
1:100_#15 | 0.63 | 22.00 |
1:100_#16 | 2.09 | 73.04 |
Sequencing library pooling[edit]
Sequencing library IDs: Initial-Project-Flow.sorting.date-# of nuclei-Experiment name-Adaptor dilution-pool #-Date
- -Pool1: NP-RRBS-Apr10-1nu-Apr#3-1to50-p1-Apr25, 2014
Sample # Index Conc. (ng/ul) Volume for 20ng (ul) 1:50_#1 2 2.10 9.52 1:50_#2 4 1.13 17.70 1:50_#3 5 0.68 29.41 1:50_#6 12 2.61 7.66
- -Pool2: NP-RRBS-Apr10-1nu-Apr#3-1to50-p2-Apr25, 2014
Sample # Index Conc. (ng/ul) Volume for 40ng (ul) 1:50_#15 2 3.24 12.35 1:50_#16 4 3.61 11.08
- -Pool3: NP-RRBS-Apr10-10nu-Apr#3-1to50-p3-Apr25, 2014
Sample # Index Conc. (ng/ul) Volume for 100ng (ul) 1:50_#13 7 7.31 13.69 1:50_#14 12 9.01 11.10
- -Pool4: NP-RRBS-Apr10-1nu-Apr#3-1to100-p4-Apr25, 2014
Sample # Index Conc. (ng/ul) Volume for 25ng (ul) 1:100_#2 4 3.15 7.94 1:100_#4 6 0.77 32.33 1:100_#5 7 1.19 20.93 1:100_#6 12 1.46 17.15
- -Pool5: NP-RRBS-Apr10-1nu-Apr#3-1to100-p5-Apr25, 2014
Sample # Index Conc. (ng/ul) Volume for 30ng (ul) 1:100_#7 2 1.00 30.00 1:100_#16 4 2.09 14.35
Pool2 File:ZhangLab 2 2014-05-06 12hr 22min pool2.jpg Pool4 File:ZhangLab 2 2014-05-06 12hr 20min pool4.jpg
- - Load 3ul of each sequencing library pool
File:ZhangLab 2 2014-05-08 19hr 54min PAGE verified.jpg
Sequencing: HL166 run PE 50+7+8
- - NP-RRBS-Apr10_1nu_Apr#3_1to50_p1-Apr25: lane 1, 50%
- - NP-RRBS-Apr10_1nu_Apr#3_1to50_p2-Apr25: lane 2, 25%
- - NP-RRBS-Apr10_10nu_Apr#3_1to50_p3-Apr25: lane 2, 25%
- - NP-RRBS-Apr10_1nu_Apr#3_1to100_p4-Apr25: lane 3, 50%
- - NP-RRBS-Apr10_1nu_Apr#3_1to100_p5-Apr25: lane 4, 25%