Noi/NOTES/2014-6-29: Difference between revisions

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* Performed bisulfite conversion using LifeTech MethylCode™ Bisulfite Conversion Kit and did the same procedure following manufacturer's instruction and elute with 38ul Elution buffer.
* Performed bisulfite conversion using LifeTech MethylCode™ Bisulfite Conversion Kit and did the same procedure following manufacturer's instruction and elute with 38ul Elution buffer.
'''''<span style="color:crimson"><u>Prep</u></span>'''''<br>
'''''<span style="color:crimson"><u>Prep</u></span>'''''<br>
:- Prepare 2 tubes of complete CT Conversion Reagent by adding 800ul H2O, 50ul Resuspension Buffer, and 300ul Dilution Buffer
:- Prepare 2 tubes of complete CT Conversion Reagent by adding 850ul H2O, 50ul Resuspension Buffer, and 300ul Dilution Buffer. If CT Conversion reagent prepare with 800ul H2O, need to add 5ul of H2O to 25ul adaptor-ligated DNA and added 120ul of CT conversion reagent.
:- Mix well and spin down the tube
:- Mix well and spin down the tube
:- Put on thermomixer at 37C for 15min, 1,000rpm
:- Put on thermomixer at 37C for 15min, 1,000rpm
Line 200: Line 200:
:- Spin down column at 14,000 rpm for 3min <u>(do not discard spnt)</u>
:- Spin down column at 14,000 rpm for 3min <u>(do not discard spnt)</u>
:- Transfer column to prepared 1.5mL LoBind tube & make sure that column does not touch spnt in the collecting tube before transferring to new tube
:- Transfer column to prepared 1.5mL LoBind tube & make sure that column does not touch spnt in the collecting tube before transferring to new tube
:- Add 38ul of Elution Buffer (warm Elution Buffer at 60C for 10min)
:- Add 30ul of Elution Buffer (warm Elution Buffer at 60C for 10min)
:- Wait for 2min
:- Wait for 2min
:- Spin down column at 12,000 rpm for 1min
:- Spin down column at 12,000 rpm for 1min
:- Continue to amplification (During elution step, PCR mix should be prepared)
:- Continue to amplification (During elution step, PCR mix should be prepared)

Revision as of 06:58, 1 July 2014

Preparation of RRBS sequencing libraries of plasma DNA from colon and lung cancer patients

Link to calendar

Sample info

Project ID Zhang lab ID Conc. (ng/ul)
UCSD-006.1 6-P_1 0.200
UCSD-006.2 6-P_2 1.100
UCSD-006.3 6-P_3 1.570
UCSD-006.4 6-P_4 0.889
UCSD-006.5 6-P_5 0.487
UCSD-006.6 6-P_6 0.249
UCSD-006.7 6-P_7 0.247
UCSD-006.8 6-P_8 0.226
UCSD-006.9 6-P_9 0.408
UCSD-006.10 6-P_10 0.333
Project ID Zhang lab ID Conc. (ng/ul)
UCSD-007.1 7-P_1 0.692
UCSD-007.2 7-P_2 1.140
UCSD-007.3 7-P_3 0.208
UCSD-007.4 7-P_4 0.202
UCSD-007.5 7-P_5 1.110
UCSD-007.6 7-P_6 0.233
UCSD-007.7 7-P_7 0.229
UCSD-007.8 7-P_8 0.167
UCSD-007.9 7-P_9 0.231
UCSD-007.10 7-P_10 0.203
  • X-Y_Z:
    • X = project ID number. 4 = pancreas, 6 = colon, 7 = lung
    • Y = sample type. T = tissue, P = plasma, S = serum
    • Z = sample ID

1) MspI digestion

  • Note that the protocol of low-input DNA (1-5ng) will be different from the STD RRBS protocol (100ng).
  • Conditions: Digest 1ng of DNA with 10U of MspI in 18ul reaction at 37C O/N (normally 3h) and heat inactivate at 65C 20min
  • For 1ng DNA input, I used 1ul (10U) of MspI.
  • I spike in 1% of unmeth lambda DNA (10pg)
  • I actually start MspI at late night of 2014-06-29, so I incubated it O/N as it doesn't hurt to incubate DNA with MspI longer than 3h.


Msp I Reaction Mix

Components 1x rxn 26 rxn Mix
MspI (10U/ul) 1.00 26.00
10x Tango Buffer 2.00 52.00
12pg/ul unmeth-lambda DNA 0.83 21.67
H2O 8.17 212.33
Total 12.00

Prep
- Aliquot 39ul of MspI reaction mix to 8-tube strip.

- Add H2O to each tube
- Add 1ng of DNA to each tube
- Add 12ul of MspI reaction Mix to each tube and pipette few times
- Spin down the tubes
- Mix by gentle pulse-vortexing on PCR rack 10x
- Spin down the tubes
- 37C, O/N (~12.5h) --> 65C, 20min --> hold at 4C (set heat lid)


2) End-repair/dA-tailing

Prep
- Mix 29ul of Klenow exo- with 29ul of dA:dC:dG (stock 20mM:2mM:2mM) just right before adding to MspI digested DNA
- Aliquot 7ul of Klenow, exo- and dA:dC:dG mix to 8-tube strip.

- Add 2ul of Klenow, exo- and dA:dC:dG mix to each tube of MspI digested DNA with multichannel pipette. Pipette few times
- Spin down the tube
- Mix by gentle pulse-vortexing on PCR rack 10x
- Spin down the tube
- 30C, 20min --> 37C, 20min --> hold at 4C (no heat lid)
- 75C, 10min --> hold at 4C (heat lid on)


3) Methylated adaptor ligation

  • Conditions: Add 1ul of 1:20 diluted TruSeq adaptors (do not know actual concentration of adaptors) in 25ul ligation reaction with 6000U of T4 DNA ligase

Ligation reaction set up

Prep
- Thaw diluted TruSeq adaptor on ice (used the diluted adaptors prepared on 2014-06-20, keep it on ice all the time)
- Prepare ligation reaction mix

Ligation reaction Mix

Components 1x rxn 26x rxn
dA-tailed DNA 20.00 0.00
10X Tango buffer 0.50 13.00
HC T4 DNA ligase (30units/ul) 1.00 26.00
10mM ATP 1.25 32.50
H2O 1.25 32.50
Total 24.00

Prep
- Aliquot 13ul of ligation reaction mix to 8-tube strip.

- Add 1ul of 1:20 diluted TruSeq adaptors and pipette few times to make sure adaptors were added to the reaction
Sample TruSeq Index Sample TruSeq Index
6-P_1 13 7-P_1 1
6-P_2 14 7-P_2 2
6-P_3 15 7-P_3 4
6-P_4 16 7-P_4 5
6-P_5 27 7-P_5 6
6-P_6 18 7-P_6 7
6-P_7 19 7-P_7 8
6-P_8 20 7-P_8 10
6-P_9 21 7-P_9 11
6-P_10 22 7-P_10 12
6-P_NTC1 22 7-P_NTC1 12
6-P_NTC2 22 7-P_NTC2 12
- Add 4ul of ligation reaction mix with multichannel pipette
- Spin down the tube
- Mix by gentle pulse-vortexing on PCR rack 10x
- Spin down the tube
- 16C, 20h (no heat lid. Ligation time is flexible. I usually do at least 20h at 16C)
- Mix by gentle pulse-vortexing on PCR rack 5x
- Quickly spin down
- Heat inactivate at 65C, 20min --> hold at 4C (heat lid on)

Bisulfite conversion

  • Performed bisulfite conversion using LifeTech MethylCode™ Bisulfite Conversion Kit and did the same procedure following manufacturer's instruction and elute with 38ul Elution buffer.

Prep

- Prepare 2 tubes of complete CT Conversion Reagent by adding 850ul H2O, 50ul Resuspension Buffer, and 300ul Dilution Buffer. If CT Conversion reagent prepare with 800ul H2O, need to add 5ul of H2O to 25ul adaptor-ligated DNA and added 120ul of CT conversion reagent.
- Mix well and spin down the tube
- Put on thermomixer at 37C for 15min, 1,000rpm
- Solution should be clear
- Spin down the tube
- Add 120ul complete CT Converson Reagent to 30ul bead-purified adaptor ligated DNA --> total volume 150ul
- Mix by pipetting 10X with multi-channel pipette
- Spin down the tube
- 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
- Add 600ul Binding Buffer to each column
- Adjust p200 to 200ul and transfer bisulfite treated DNA to the column
- Mix by pipetting 5x
- Spin down column at 14,000 rpm for 30s
- Discard spnt
- Add 100ul Wash Buffer (Aliquot Wash Buffer in 5mL tube)
- Spin down column at 14,000 rpm for 30s (do not discard spnt)
- Add 200ul Desulfonation Buffer (aliquot Desulfonation Buffer in 2mL or 5mL tube)
- Spin down column at 14,000 rpm for 30s (do not discard spnt)
- Add 200ul Wash Buffer
- Spin down column at 14,000 rpm for 30s
- Discard spnt
- Add 200ul Wash Buffer
- Spin down column at 14,000 rpm for 3min (do not discard spnt)
- Transfer column to prepared 1.5mL LoBind tube & make sure that column does not touch spnt in the collecting tube before transferring to new tube
- Add 30ul of Elution Buffer (warm Elution Buffer at 60C for 10min)
- Wait for 2min
- Spin down column at 12,000 rpm for 1min
- Continue to amplification (During elution step, PCR mix should be prepared)