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

From ZhangLabWiki
Jump to navigation Jump to search
>Noi
(Created page with "= Preparation of RRBS sequencing libraries of plasma DNA from colon and lung cancer patients = '''Link to calendar''' ==== Sample info ====")
 
>Noi
mNo edit summary
Line 2: Line 2:
[[noi:DMR220k_LabNotes#2014| '''Link to calendar''']]
[[noi:DMR220k_LabNotes#2014| '''Link to calendar''']]
==== Sample info ====
==== Sample info ====
{| {{table}} class = wikitable
| align="center" style="width:120px;background:#f0f0f0;"|'''Project ID'''
| align="center" style="width:80px;background:#f0f0f0;"|'''Zhang lab ID'''
| align="center" style="width:80px;background:#f0f0f0;"|'''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
|}
{| {{table}} class = wikitable
| align="center" style="width:120px;background:#f0f0f0;"|'''Project ID'''
| align="center" style="width:80px;background:#f0f0f0;"|'''Zhang lab ID'''
| align="center" style="width:80px;background:#f0f0f0;"|'''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.<br>
<br>
==== <u>Msp I Reaction Mix</u> ====
{| {{table}} border = 1
| align="center" style="background:#f0f0f0;"|'''Components'''
| align="center" style="width:80px;background:#f0f0f0;"|'''1x rxn'''
| align="center" style="width:80px;background:#f0f0f0;"|'''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||
|}
'''''<span style="color:crimson"><u>Prep</u></span>'''''<br>
- 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)<br>
<br>
== 2) End-repair/dA-tailing ==
'''''<span style="color:crimson"><u>Prep</u></span>'''''<br>
- Mix 29ul of Klenow exo- with 29ul of dA:dC:dG (stock 20mM:2mM:2mM) just right before adding to MspI digested DNA<br>
- 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)
<br>
== 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
==== <u>Ligation reaction set up</u> ====
'''''<span style="color:crimson"><u>Prep</u></span>'''''<br>
- Thaw diluted TruSeq adaptor on ice (used the diluted adaptors prepared on 2014-06-20, keep it on ice all the time)<br>
- Prepare ligation reaction mix
====<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;"|'''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||
|}
'''''<span style="color:crimson"><u>Prep</u></span>'''''<br>
- 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
:{| {{table}} class = wikitable
| align="center" style="width:90px;background:#f0f0f0;"|'''Sample'''
| align="center" style="width:70px;background:#f0f0f0;"|'''TruSeq Index'''
| align="center" style="width:90px;background:#f0f0f0;"|'''Sample'''
| align="center" style="width:70px;background:#f0f0f0;"|'''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_NTC||22||7-P_NTC||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.
'''''<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
:- 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
:- <u>Discard spnt</u>
:- Add 100ul Wash Buffer (Aliquot Wash Buffer in 5mL tube)
:- Spin down column at 14,000 rpm for 30s <u>(do not discard spnt)</u>
:- Add 200ul 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 down column at 14,000 rpm for 30s
:- <u>Discard spnt</u>
:- Add 200ul Wash Buffer
:- 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
:- Add 38ul 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)

Revision as of 19:14, 30 June 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_NTC 22 7-P_NTC 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 800ul H2O, 50ul Resuspension Buffer, and 300ul Dilution Buffer
- 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 38ul 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)