Noi/NOTES/2014-4-23

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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
- 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
- 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
File:ZhangLab 2 2014-04-25 17hr 49min beadpurified positive 6-9-10-11-13-14 5ul.jpg

Discussion[edit]

File:ZhangLab 2 2014-04-27 Apr