Noi/NOTES/2014-6-16: Difference between revisions
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:- 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 | :- Add 15ul of MspI reaction Mix to each tube and pipette few times | ||
:- Mix by | :- Spin down the tubes | ||
:- Spin down the | :- 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 | :- 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 | - 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 | :- 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 | :- 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)