Noi/NOTES/2014-6-16: Difference between revisions

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Line 90: Line 90:
:- Add H2O to each tube
:- Add H2O to each tube
:- Add 100ng of DNA to each tube
:- Add 100ng of DNA to each tube
:- Add 15ul of MspI reaction Mix to each tube and pipette 5x
:- Add 15ul of MspI reaction Mix to each tube and pipette few times
:- Mix by gently vortexing on PCR rack 10x
:- Spin down the tubes
:- Spin down the tube
:- Mix by gentle pulse-vortexing on PCR rack 10x
:- Spin down the tubes
:- 37C, 3h --> 65C, 20min --> hold at 4C (set heat lid)
:- 37C, 3h --> 65C, 20min --> hold at 4C (set heat lid)
=== 2) End-repair/dA-tailing ===
=== 2) End-repair/dA-tailing ===
Line 98: Line 99:
- Mix 13ul of Klenow exo- with 13ul of dA:dC:dG (stock 20mM:2mM:2mM) just right before adding to MspI digested DNA
- Mix 13ul of Klenow exo- with 13ul of dA:dC:dG (stock 20mM:2mM:2mM) just right before adding to MspI digested DNA
:- Add 2ul of Klwnow, exo- and dA:dC:dG mix to each tube of MspI digested DNA. Pipetting few times
:- Add 2ul of Klwnow, exo- and dA:dC:dG mix to each tube of MspI digested DNA. Pipetting few times
:- Mix by gently vortexing on PCR rack 10x
:- Mix by gentle pulse-vortexing on PCR rack 10x
:- Spin down the tube
:- Spin down the tube
:- 30C, 20min --> 37C, 20min --> hold at 4C  (no heat lid)
:- 30C, 20min --> 37C, 20min --> hold at 4C  (no heat lid)
Line 104: Line 105:
=== 3) Methylated adaptor ligation ===
=== 3) Methylated adaptor ligation ===
* Conditions: Add 1ul of non-diluted TruSeq adaptors (do not know actual concentration of adaptors) in 30ul ligation reaction with 6000U of T4 DNA ligase
* Conditions: Add 1ul of non-diluted TruSeq adaptors (do not know actual concentration of adaptors) in 30ul ligation reaction with 6000U of T4 DNA ligase
* Before set up ligation reaction, purify end-repaired DNA with 2x volume AMPure beads
* Before set up ligation reaction, purify end-repaired DNA with 2x volume AMPure beads (64ul each) and resuspend the bead with 20ul EB buffer
==== <u>AMPure bead purification</u> ====
==== <u>AMPure bead purification</u> ====
'''''<span style="color:crimson"><u>Prep</u></span>'''''<br>
'''''<span style="color:crimson"><u>Prep</u></span>'''''<br>
- Freshly prepare 5mL of 75% EtOH by mixing 3.75mL of 100% EtOH with 1.25mL of H2O (prepare when the tubes sit on magnet)<br>
- Freshly prepare 5mL of 75% EtOH by mixing 3.75mL of 100% EtOH with 1.25mL of H2O (prepare when the tubes sit on magnet)<br>
- Aliquot 360ul pf AMPure beads in 1.5mL tube and leave at RT<br>
- Aliquot 360ul of AMPure beads in 1.5mL tube and leave at RT<br>
:- Add 64ul AMPure bead. Mix by pipetting 10x
:- Add 64ul AMPure bead. Mix by pipetting 10x
:- Wait for 30min
:- Wait for 30min
Line 142: Line 143:
:- Add 1ul of non-diluted TruSeq adaptors and pipette few times to make sure adaptors were added to the reaction
:- Add 1ul of non-diluted TruSeq adaptors and pipette few times to make sure adaptors were added to the reaction
:- Add 9ul of ligation reaction mix
:- Add 9ul of ligation reaction mix
:- Mix by gently vortexing on PCR rack 10x
:- Spin down the tube
:- Mix by gentle pulse-vortexing on PCR rack 10x
:- Spin down the tube
:- Spin down the tube
:- 16C, 20h (no heat lid)
:- 16C, 20h (no heat lid)
:- Mix by gently vortexing on PCR rack 5x  
:- Mix by gentle pulse-vortexing on PCR rack 5x
:- Quickly spin down
:- Quickly spin down
:- Heat inactivate at 65C, 20min --> hold at 4C (heat lid on)
:- Heat inactivate at 65C, 20min --> hold at 4C (heat lid on)
* Before continue to bisulfite conversion, purify adaptor-ligated DNA with 2x volume AMPure beads (60ul each) and resuspend the bead with 31ul EB buffer
==== <u>AMPure bead purification</u> ====
'''''<span style="color:crimson"><u>Prep</u></span>'''''<br>
- Freshly prepare 5mL of 75% EtOH by mixing 3.75mL of 100% EtOH with 1.25mL of H2O (prepare when the tubes sit on magnet)<br>
- Aliquot 335ul of AMPure beads and 335ul of 20% PEG 8000/5M NaCl in 1.5mL tube, mix well and leave at RT<br>
:- Add 60ul AMPure bead. Mix by pipetting 10x
:- Wait for 30min
:- Transfer to sit on magnet for 5min
:- Wash twice with 180ul freshly prepared 75% EtOH
::- After adding 75% EtOH (1st time), wait for 30sec before pipetting up & down 5x
:- Remove all 75% EtOH
:- Dry the bead for 20min
:- Resuspend with 31 EB Buffer  and transfer 30ul of adaptor-ligated DNA to new strip tubes <u>(discard the beads)</u>
:- Continue to bisulfite conversion
=== 3) Bisulfite conversion ===
* Performed bisulfite conversion using the same procedure following manufacturer's instruction [[http://tools.lifetechnologies.com/content/sfs/manuals/methylcode_bisulfite_man.pdf]] and elute with 38ul Elution buffer.
'''''<span style="color:crimson"><u>Prep</u></span>'''''<br>
- UV H2O <br>
- Label 1.5mL LoBind tube & UV<br>
- Resuspend CT Conversion Reagent (during drying the beads)
:- For 30ul sample: Add 800ul H2O, 50ul Suspension Buffer, and 300ul Dilution Buffer
:- Mix well and spin down the tube
:- Put on thermomixer at 37C for 15min, 1000rpm
:- Solution should be clear
:- Spin down the tube
------------------
:- Add 120ul complete CT Converson Reagent to 30ul bead-purified adaptor ligated DNA
:- 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
:- Spin down column at 14,000 rpm for 30s
:- <u>Discard spnt</u>
:- Add 100ul Wash Buffer (Aliquot Wash Buffer in 5mL tube)
:- Spin column at 14,000 rpm for 30s <u>(do not discard spnt)</u>
:- Add 20ul Desulfonation Buffer (aliquot Desulfonation Buffer in 2mL or 5mL tube)
:- Spin down column at 14,000 rpm for 30s  <u>(do not discard spnt)</u>
:- Add 200ul Wash Buffer
:- Spin column at 14,000 rpm for 30s
:- <u>Discard spnt</u>
:- Add 200ul Wash Buffer
:- Spin column at 14,000 rpm for 3min <u>(do not discard spnt)</u>

Revision as of 15:18, 16 June 2014

Preparation of RRBS sequencing libraries of solid tumor tissue sample from pancreatic cancer patients

Sample info

Project ID Zhang lab ID Conc. (ng/ul)
UCSD-004-04 PC-T-1 82.70
UCSD-004-05 PC-T-2 35.80
UCSD-004-07 PC-T-4 42.80
UCSD-004-09 PC-T-6 15.20
UCSD-004-10 PC-T-7 16.30

1) MspI digestion

  • Conditions: Digest 100ng of DNA with 20U of MspI in 30ul reaction at 37C 3h and heat inactivate at 65C 1h

MspI digestion set up

Strip1 PC-T-1_r1 PC-T-1_r2 PC-T-2_r1 PC-T-2_r2 PC-T-4_r1 PC-T-4_r2 PC-T-6_r1 PC-T-6_r2
DNA conc. (ng/ul) 82.70 82.70 35.80 35.80 42.80 42.80 15.20 15.20
Volume for 100ng (ul) 1.21 1.21 2.79 2.79 2.34 2.34 6.58 6.58
H2O (ul) 12.96 12.96 11.38 11.38 11.83 11.83 7.59 7.59
10X Tango Buffer 3.00 3.00 3.00 3.00 3.00 3.00 3.00 3.00
MspI 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00
12pg/ul unmeth-lambda DNA 0.83 0.83 0.83 0.83 0.83 0.83 0.83 0.83
H2O in MspI Mix 10.00 10.00 10.00 10.00 10.00 10.00 10.00 10.00
Total (ul) 30.00 30.00 30.00 30.00 30.00 30.00 30.00 30.00
Strip2 PC-T-7_r1 PC-T-7_r2 NTC
DNA conc. (ng/ul) 16.30 16.30 0.00
Volume for 100ng (ul) 6.13 6.13 0.00
H2O (ul) 8.04 8.04 14.17
10X Tango Buffer 3.00 3.00 3.00
MspI 2.00 2.00 2.00
12pg/ul unmeth-lambda DNA 0.83 0.83 0.83
H2O in MspI Mix 10.00 10.00 10.00
Total (ul) 30.00 30.00 30.00

Prep
- Label strip tube for reaction set up & UV
- Aliquot H2O in 1.5mL tube & UV
- UV 1.5mL tube for MspI reaction Mix
- Thaw tumor DNA and 12pg/ul unmeth-lambda DNA
- All buffers in this experiment are aliquoted in 8-tube strip for single-use to avoid contamination

Msp I reaction Mix

Components 1x rxn 9.5 rxn Mix
MspI (10U/ul) 2.00 19.00
10x Tango Buffer 3.00 28.50
H2O 10.00 95.00
Total 15.00
- Add H2O to each tube
- Add 100ng of DNA to each tube
- Add 15ul 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, 3h --> 65C, 20min --> hold at 4C (set heat lid)

2) End-repair/dA-tailing

Prep
- Mix 13ul of Klenow exo- with 13ul of dA:dC:dG (stock 20mM:2mM:2mM) just right before adding to MspI digested DNA

- Add 2ul of Klwnow, exo- and dA:dC:dG mix to each tube of MspI digested DNA. Pipetting few times
- 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 non-diluted TruSeq adaptors (do not know actual concentration of adaptors) in 30ul ligation reaction with 6000U of T4 DNA ligase
  • Before set up ligation reaction, purify end-repaired DNA with 2x volume AMPure beads (64ul each) and resuspend the bead with 20ul EB buffer

AMPure bead purification

Prep
- Freshly prepare 5mL of 75% EtOH by mixing 3.75mL of 100% EtOH with 1.25mL of H2O (prepare when the tubes sit on magnet)
- Aliquot 360ul of AMPure beads in 1.5mL tube and leave at RT

- Add 64ul AMPure bead. Mix by pipetting 10x
- Wait for 30min
- Transfer to sit on magnet for 5min
- Wash twice with 180ul freshly prepared 75% EtOH
- After adding 75% EtOH (1st time), wait for 30sec before pipetting up & down 5x
- Remove all 75% EtOH
- Dry the bead for 20min
- Resuspend with 20 EB Buffer (keep the beads in the tubes!)

Ligation reaction set up

Prep
- Thaw TruSeq adaptor on ice (during drying the beads, keep it on ice all the time)
- Prepare ligation reaction mix (during drying the beads)

Ligation reaction Mix

Components 1x rxn 12rxn Mix
dA-tailed DNA 20.00 0.00
10x Ligation Buffer 3.00 36.00
T4 DNA Ligase (6000U/ul) 1.00 12.00
TruSeq Adaptor DNA 1.00 0.00
H2O 5.00 60.00
Total 30.00
- Add 1ul of non-diluted TruSeq adaptors and pipette few times to make sure adaptors were added to the reaction
- Add 9ul of ligation reaction mix
- Spin down the tube
- Mix by gentle pulse-vortexing on PCR rack 10x
- Spin down the tube
- 16C, 20h (no heat lid)
- Mix by gentle pulse-vortexing on PCR rack 5x
- Quickly spin down
- Heat inactivate at 65C, 20min --> hold at 4C (heat lid on)
  • Before continue to bisulfite conversion, purify adaptor-ligated DNA with 2x volume AMPure beads (60ul each) and resuspend the bead with 31ul EB buffer

AMPure bead purification

Prep
- Freshly prepare 5mL of 75% EtOH by mixing 3.75mL of 100% EtOH with 1.25mL of H2O (prepare when the tubes sit on magnet)
- Aliquot 335ul of AMPure beads and 335ul of 20% PEG 8000/5M NaCl in 1.5mL tube, mix well and leave at RT

- Add 60ul AMPure bead. Mix by pipetting 10x
- Wait for 30min
- Transfer to sit on magnet for 5min
- Wash twice with 180ul freshly prepared 75% EtOH
- After adding 75% EtOH (1st time), wait for 30sec before pipetting up & down 5x
- Remove all 75% EtOH
- Dry the bead for 20min
- Resuspend with 31 EB Buffer and transfer 30ul of adaptor-ligated DNA to new strip tubes (discard the beads)
- Continue to bisulfite conversion

3) Bisulfite conversion

  • Performed bisulfite conversion using the same procedure following manufacturer's instruction [[1]] and elute with 38ul Elution buffer.

Prep
- UV H2O
- Label 1.5mL LoBind tube & UV
- Resuspend CT Conversion Reagent (during drying the beads)

- For 30ul sample: Add 800ul H2O, 50ul Suspension Buffer, and 300ul Dilution Buffer
- Mix well and spin down the tube
- Put on thermomixer at 37C for 15min, 1000rpm
- Solution should be clear
- Spin down the tube

- Add 120ul complete CT Converson Reagent to 30ul bead-purified adaptor ligated DNA
- 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
- Spin down column at 14,000 rpm for 30s
- Discard spnt
- Add 100ul Wash Buffer (Aliquot Wash Buffer in 5mL tube)
- Spin column at 14,000 rpm for 30s (do not discard spnt)
- Add 20ul 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 column at 14,000 rpm for 30s
- Discard spnt
- Add 200ul Wash Buffer
- Spin column at 14,000 rpm for 3min (do not discard spnt)