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>Noi (Created page with "= Preparation of RRBS sequencing libraries of solid tumor tissue sample from pancreatic cancer patients = ==== Sample info ==== {| {{table}} class = wikitable | align="center"...") |
>Noi mNo edit summary |
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| UCSD-004-10||PC-T-7||16.30 | | UCSD-004-10||PC-T-7||16.30 | ||
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=== MspI digestion === | === 1) MspI digestion === | ||
* Conditions: Digest 100ng of DNA with 20U of MspI in 30ul reaction at 37C 3h and heat inactivate at 65C 1h | * 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 ==== | ==== <u>MspI digestion set up</u> ==== | ||
{| {{table}} class = wikitable | {| {{table}} class = wikitable | ||
| align="center" style="width:140px;background:#f0f0f0;"|'''Strip1''' | | align="center" style="width:140px;background:#f0f0f0;"|'''Strip1''' | ||
Line 65: | Line 65: | ||
|} | |} | ||
'''''<span style="color:crimson"><u>Prep</u></span>'''''<br> | '''''<span style="color:crimson"><u>Prep</u></span>'''''<br> | ||
- UV strip tube for reaction set up<br> | |||
- Aliquot H2O in 1.5mL tube & UV<br> | |||
- UV 1.5mL tube for MspI reaction Mix | |||
==== <u>Msp I reaction Mix</u> ==== | ==== <u>Msp I reaction Mix</u> ==== | ||
{| {{table}} border = 1 | {| {{table}} border = 1 | ||
Line 82: | Line 82: | ||
| Total||15.00|| | | Total||15.00|| | ||
|} | |} | ||
:- Add H2O to each tube | :- Add H2O to each tube | ||
:- Add 100ng of DNA to each tube | :- Add 100ng of DNA to each tube | ||
Line 88: | Line 87: | ||
:- Mix by gently vortexing on PCR rack 10x | :- Mix by gently vortexing on PCR rack 10x | ||
:- Spin down the tube | :- Spin down the tube | ||
:- | :- 37C, 3h --> 65C, 20min --> hold at 4C (set heat lid) | ||
=== 2) End-repair/dA-tailing === | |||
'''''<span style="color:crimson"><u>Prep</u></span>'''''<br> | |||
- 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 gently 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 | |||
==== <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 (when the bead sit on magnet)<br> | |||
- Aliquot 360ul pf AMPure beads in 1.5mL tube and leave at RT<br> | |||
:- Add 64ul AMPure bead. Mix by pipetting 10x | |||
:- Sit for 30min | |||
:- Transfer to sit on magnet for 5min | |||
:- Wash twice with 180ul freshly prepared 80% EtOH | |||
:- Dry the bead for 20min | |||
:- Resuspend with 20 EB Buffer <u>''(keep the beads in the tubes!)''</u> | |||
==== <u>Ligation reaction set up</u> ==== | |||
'''''<span style="color:crimson"><u>Prep</u></span>'''''<br> | |||
- Thaw TruSeq adaptor on ice (during drying the beads, keep it on ice all the time)<br> | |||
- Prepare ligation reaction mix (during drying the beads) | |||
====<u> Ligation reaction Mix</u> ==== | |||
:{| {{table}} border = 1 | |||
| align="center" style="width:140px;background:#f0f0f0;"|'''Components''' | |||
| align="center" style="width:70px;background:#f0f0f0;"|'''1x rxn''' | |||
| align="center" style="width:70px;background:#f0f0f0;"|'''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 | |||
:- Mix by gently vortexing on PCR rack 10x | |||
:- Spin down the tube | |||
:- 16C, 20h (no heat lid) | |||
:- Mix by gently vortexing on PCR rack 5x | |||
:- Quickly spin down | |||
:- Heat inactivate at 65C, 20min --> hold at 4C (heat lid on) |
Revision as of 09:08, 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) | 13.79 | 13.79 | 12.21 | 12.21 | 12.66 | 12.66 | 8.42 | 8.42 |
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 |
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) | 8.87 | 8.87 | 15.00 |
10X Tango Buffer | 3.00 | 3.00 | 3.00 |
MspI | 2.00 | 2.00 | 2.00 |
H2O in MspI Mix | 10.00 | 10.00 | 10.00 |
Total (ul) | 30.00 | 30.00 | 30.00 |
Prep
- UV strip tube for reaction set up
- Aliquot H2O in 1.5mL tube & UV
- UV 1.5mL tube for MspI reaction Mix
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 5x
- - Mix by gently vortexing on PCR rack 10x
- - Spin down the tube
- - 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 gently 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
AMPure bead purification
Prep
- Freshly prepare 5mL of 75% EtOH by mixing 3.75mL of 100% EtOH with 1.25mL of H2O (when the bead sit on magnet)
- Aliquot 360ul pf AMPure beads in 1.5mL tube and leave at RT
- - Add 64ul AMPure bead. Mix by pipetting 10x
- - Sit for 30min
- - Transfer to sit on magnet for 5min
- - Wash twice with 180ul freshly prepared 80% 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
- - Mix by gently vortexing on PCR rack 10x
- - Spin down the tube
- - 16C, 20h (no heat lid)
- - Mix by gently vortexing on PCR rack 5x
- - Quickly spin down
- - Heat inactivate at 65C, 20min --> hold at 4C (heat lid on)