Noi/NOTES/2014-4-25

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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
- 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

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

File:Aprn03 gel2 e.jpg

File:Aprno3 gel1.jpg

File:Aprno3 gel3.jpg

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%