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

From ZhangLabWiki
Jump to navigation Jump to search
>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
Line 16: Line 16:
| UCSD-004-10||PC-T-7||16.30
| UCSD-004-10||PC-T-7||16.30
|}
|}
=== 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
- UV strip tube for reaction set up<br>
: - Aliquot H2O in 1.5mL tube & UV
- Aliquot H2O in 1.5mL tube & UV<br>
:- UV 1.5mL tube for MspI reaction Mix
- 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||
|}
|}
==== <u>Procedures</u> ====
:- 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
:- In program '''Noi: MspI''': 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 ===
'''''<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)