Noi/NOTES/2014-4-21: Difference between revisions

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= scRRBS experiment Apr#1 =
= scRRBS experiment: <span style="color:mediumblue">Apr#1</span> =
* I stopped doing scRRBS experiment for a while since I never get it work for single nuclei, and I think I should use the same barcoded adaptors as used by Dr. Tang group.<br>
'''Link to calendar: [[http://genome-tech.ucsd.edu/LabNotes/index.php/noi:DMR220k_LabNotes#2014]]'''
* I stopped doing scRRBS experiment for a while since I never get it works for single nuclei, and I think I should use the same barcoded methylated adaptors as used by Dr. Tang's group.<br>
* I found an old TrueSeq DNA Sample Prep kit in (Set A, part # 15013178) -20C. I then wanted to try these barcoded adaptors for ssRRBS while waiting for TrueSeq kit requested by Dr. Zhang. In this kit, I found only 6 indexed adaptors, including Ind2, Ind 4, Ind 5, Ind 6, Ind7 and Ind 12. <br>
* I found an old TrueSeq DNA Sample Prep kit in (Set A, part # 15013178) -20C. I then wanted to try these barcoded adaptors for ssRRBS while waiting for TrueSeq kit requested by Dr. Zhang. In this kit, I found only 6 indexed adaptors, including Ind2, Ind 4, Ind 5, Ind 6, Ind7 and Ind 12. <br>
* I have contacted with Dr. Tang's group for more information of scRRBS protocol in very details, and found there are many things I did different from them. To be able to repeat the protocol and get it work for single cell, I followed everything noted by them.<br>
* I have contacted with Dr. Tang's group for more information of scRRBS protocol in very details, and found there are many things I did differently from them. To be able to repeat the protocol and get it works for single cell, I followed everything noted by them.<br>
== More information from Dr. Tang's group ==
== More information from Dr. Tang's group ==
* They used 80% of bead purified (two-round purification) 1st round amplicons as the template for the 2nd round PCR.
* They used 80% of bead purified (two-round purification) 1st round amplicons as the template for the 2nd round PCR.
* In both the 1st round and the 2nd round PCR, they did not do qPCR to monitor amplicons. They said that that the bisulfite-treated DNA was extremely low, so they did not monitor it.
* In both the 1st round and the 2nd round PCR, <span style="color:red">they did not do qPCR to monitor amplicons</span>. They said that that the bisulfite-treated DNA was extremely low, so they did not monitor it.
: - '''I think this might be the key that I never get success in single-nuclei experiment as I alway stopped reaction during the 2nd round PCR earlier before reaching 22 cycles (like 12 or 15 based on qPCR curve)'''
: - '''<span style="color:blue">I think this might be the key reason that I never get success in single-nuclei experiment as I alway stopped reaction during the 2nd round PCR earlier before reaching 22 cycles (between 12-15 based on qPCR curve)</span>'''
* They heat-inactivate after MspI digestion, end-repair/dA-tailing, and ligation
* They heat-inactivate after ''Msp''I digestion, end-repair/dA-tailing, and methylated adaptors ligation
: - MspI (not specify)
: - ''Msp''I (not specify)
: - End-repair/dA-tailing (I did 75C, 20min
: - End-repair/dA-tailing (I did 75C, 20min
: - Ligation (65C, 20min) --> from Gu et al, 2011 (Nature Protocol). They suggest not to heat the lid since I could potentially destroy T4 DNA ligase.
: - Ligation (65C, 20min) --> from Gu et al, 2011 (Nature Protocol). They suggest not to heat the lid since it could potentially destroy T4 DNA ligase.
* They incubate ligation reaction at 16C, 30min --> 4C (at least 8hr). I previously incubate at 16C for at least 12h.
* They incubate ligation reaction at 16C, 30min --> 4C (at least 8hr). I previously incubate at 16C for at least 12h.
* I share all of information these info with Yun Liu, the postdoc fellow in Dr. Feinberg group, since he wants to try this protocol too.
* For PCR, thy used 200uM of dNTP and 300nM of primer-pair
* I share all of these info with Yun Liu, the postdoctoral fellow in Dr. Feinberg's group, since he wants to try this protocol too.


----
----
=== NOTE ===
* I used flow-sorted nuclei on <u>2014-04-10</u> for this experiment.
* I used flow-sorted nuclei on <u>2014-04-10</u> for this experiment.
* I still skip unmethylated lambda DNA since I want to avoid the background in trial experiment. Once the protocol is settle, I will include it to see bisulfite conversion rate
* I still skipped unmethylated lambda DNA since I want to avoid the background in trial experiment. Once the protocol is settle, I will include it to see bisulfite conversion rate
* I increased primer concentration in both 1st round and 2nd round PCR to 0.3mM or 300uM
<br>
 
== Experimental Procedures ==
==== Plate layout ====
==== Plate layout ====
{| {{table}} class = wikitable
:{| {{table}} class = wikitable
| align="center" style="background:#f0f0f0;" width="50"|''''''
| align="center" style="background:#f0f0f0;" width="50"|''''''
| align="center" style="background:#f0f0f0;" width="50"|'''1'''
| align="center" style="background:#f0f0f0;" width="50"|'''1'''
Line 41: Line 48:
|}
|}
* I include many of 0 nuclei since I want to see the consistency in all of them.
* I include many of 0 nuclei since I want to see the consistency in all of them.
=== Cell lysis ===
* Some steps were not the same as Dr. Tang'g group suggestion since I got his email after I started experiment.
:- Thaw nuclei from -80C & spin down at 2000rpm (96-well plate rotor, 5min)
 
:- Mix protease and 1.2 pg/ul unmethylated lambda DNA 1ul:0.05 ratio (30ul + 1.5ul). This will be used 1.05ul for nuclei lysis.
=== 1) Cell lysis ===
:- Add 1.05ul of protease/unmethylated lambda DNA mix
'''''<span style="color:crimson"><u>Prep</u></span>'''''<br>
- Thaw nuclei from -80C & spin down at 2,000rpm for 5min (96-well plate rotor, 5min)
:- Add 1ul of protease
:- Spin down the plate at 2,000rpm for 2min
:- Mix by gentle pulse-vortexting for 10x
:- Spin down the plate at 2,000rpm for 3min
::- I tried to avoid pipetting up and down to mix the reaction to prevent nuclei/DNA lost.  
::- I notice solution was mixed well and no foaming generated
::- I spin down the plate before and after mixing quite long to make sure that all reagents were collected to the bottom of the well
:- ''Incubate at 50C for 3hr''<br>
:- ''Incubate at 50C for 3hr''<br>
:- ''Heat inactivate at 75C for 30min''
:- ''Heat inactivate at 75C for 30min''
:- Set program to hold at 15C
:- Spin down the plate at 2,000rpm for 1min before continuing to next step
<br>
=== 2) DNA fragmentation with ''Msp''I ===
* Incubated released naked DNA with 9units of ''Msp''I in 18ul reaction at 37C for 3hr.
'''''<span style="color:crimson"><u>Prep</u></span>'''''<br>
- Prepare ''Msp''I reaction mix
:{| {{table}} class = wikitable
| align="center" style="background:#f0f0f0;"|'''Components'''
| align="center" style="background:#f0f0f0;"|'''Volume (ul)'''
| align="center" style="background:#f0f0f0;"|'''26x rxn mix'''
|-
| Lysed nuclei||5.00||0.00
|-
| 10X Tango buffer||2.00||52.00
|-
| ''Msp''I||0.90||23.40
|-
| H2O||10.10||262.60
|-
| Total||18.00||338.00
|}
- Aliquot 42ul of ''Msp''I enzyme mix to each tube in 8-tube strip
:- Add 13ul to each well with multi-channel pipette
:- Spin down the plate at 2,000rpm for 2min
:- Mix by gentle pulse-vortexting for 10x
:- Spin down the plate at 2,000rpm for 3min
:- ''Incubate at 37C for 3hr''
:- Set program to hold at 15C
:- Spin down the plate at 2,000rpm for 1min before continuing to next step
<br>
<br>
=== 3) Gap-filling/dA-tailing ===
* Add '''5 units of Klenow fragment exo-''', supplemented with '''1mM dATP, 0.1 mM dGTP and 0.1 mM of dCTP''' in 20ul reaction. (Skip dTTP because enzyme cleaves C^CGG)
'''''<span style="color:crimson"><u>Prep</u></span>'''''<br>
- Aliquot 5ul of dA:dC:dG mix (20mM:2mM:2mM) to each tube of 8-tube strip<br>
- Aliquot 3.5ul of Klenow fragment exo- to each tube of 8-tube strip<br>
:- Add 1ul of dA:dC:dG solution mix to each well with multichannel pipette
:- Add 1ul of Klenow fragment exo- to each well with multichannel pipette
:- Add 13ul to each well with multi-channel pipette
:- Spin down the plate at 2,000rpm for 2min
:- Mix by gentle pulse-vortexting for 10x
:- Spin down the plate at 2,000rpm for 3min
::- I prefer to add reagents with multichannel pipette since I want to make sure no samples were missing during adding reagents and save a lot of my time. Indeed, less pipetting can avoid contamination.
:- ''Incubate at 30C for 20min'' (for gap-filling) --> 37C for 20min (for extra dA-tailing)
:- ''Heat inactivate enzyme at 75C for 10min''
:- Set program to hold at 4C
:- Spin down the plate at 2,000rpm for 1min before continuing to next step
<br>
=== 4) Methylated adaptor ligation ===
* Ligate A-tailed DNA with 1ul of 1:20 diluted Illumina indexed methylated adaptor in total reaction 25ul at 16C for 30min and 4C for at least 8h (I did 14.5h --> next time will fix incubation time at 4C for consistency).
'''Index list'''<br>
- I have only 6 indexes, I put Ind_2 and Ind_4 twice in 8-tube strip
{| {{table}} class = wikitable
| align="center" style="background:#f0f0f0;"|'''Tube_1'''
| align="center" style="background:#f0f0f0;"|'''Tube_2'''
| align="center" style="background:#f0f0f0;"|'''Tube_3'''
| align="center" style="background:#f0f0f0;"|'''Tube_4'''
| align="center" style="background:#f0f0f0;"|'''Tube_5'''
| align="center" style="background:#f0f0f0;"|'''Tube_6'''
| align="center" style="background:#f0f0f0;"|'''Tube_7'''
| align="center" style="background:#f0f0f0;"|'''Tube_8'''
|-
| Ind_2||Ind_4||Ind_5||Ind_6||Ind_7||Ind_12||Ind_2||Ind_4
|}
'''''<span style="color:crimson"><u>Prep</u></span>'''''<br>
- Mix 1ul of indexed methylated adaptors with 20ul H2O (on ice box)<br>
- Prepare ligation reaction mix
{| {{table}} class =wikitable
| align="center" style="background:#f0f0f0;"|'''Components'''
| align="center" style="background:#f0f0f0;"|'''Volume (ul)'''
| align="center" style="background:#f0f0f0;"|'''28x rxn mix'''
|-
| dA-tailed reaction||20.00||0.00
|-
| 10X Tango buffer||0.50||14.00
|-
| HC T4 DNA ligase (30units/ul)||1.00||28.00
|-
| 10mM ATP ||1.25||35.00
|-
| H2O||1.25||35.00
|-
| Total||24.00||112.00
|}
- Aliquot 14ul of Ligation mix to each tube in 8-tube strip
:- Add 1ul of diluted methylated adapter
:- Add 4ul of ligation reaction mix
:- Spin down the plate at 2000rpm for 2min
:- Mix by gentle pulse-vortexting for 10x
:- Spin down the plate at 2,000rpm for 3min
:- ''Incubate at 16C for 30min -> 4C for 14.5h (no heat lid)''
:- ''Heat inactivate at 65C for 20min''
:- Spin down the plate at 2,000rpm for 1min before continuing to next step
:{| {{table}} class = wikitable
| align="center" style="background:#f0f0f0;"|'''Well ID'''
| align="center" style="background:#f0f0f0;"|'''Index'''
| align="center" style="background:#f0f0f0;"|'''Sample #'''
| align="center" style="background:#f0f0f0;"|'''Well ID'''
| align="center" style="background:#f0f0f0;"|'''Index'''
| align="center" style="background:#f0f0f0;"|'''Sample #'''
| align="center" style="background:#f0f0f0;"|'''Well ID'''
| align="center" style="background:#f0f0f0;"|'''Index'''
| align="center" style="background:#f0f0f0;"|'''Sample #'''
|-
| A1||Ind_2||#1||A2||Ind_2||#9||A3||Ind_2||#17
|-
| B1||Ind_4||#2||B2||Ind_4||#10||B3||Ind_4||#18
|-
| C1||Ind_5||#3||C2||Ind_5||#11||C3||Ind_5||#19
|-
| D1||Ind_6||#4||D2||Ind_6||#12||D3||Ind_6||#20
|-
| E1||Ind_7||#5||E2||Ind_7||#13||E3||Ind_7||#21
|-
| F1||Ind_12||#6||F2||Ind_12||#14||F3||Ind_12||#22
|-
| G1||Ind_2||#7||G2||Ind_2||#15||G3||Ind_2||#23
|-
| H1||Ind_4||#8||H2||Ind_4||#16||H3||Ind_4||#24
|}
'''2014-04-22'''
=== 5) Bisulfite conversion ===
* I performed bisulfite conversion using the same procedure following manufacturer's instruction and elute with 31ul elution buffer.
'''''<span style="color:crimson"><u>Prep</u></span>'''''<br>
- Prepare CT Conversion Reagent, by adding 850ul H2O, 50ul of Resuspension Buffer, and 300ul of Dilution Buffer to CT Conversion Reagent (for 25ul DNA sample --> reduce H2O from 900 to 850)
:- Add125ul of  complete CT Conversion Reagent to adaptor ligated DNA (no sample transfer to the new tube)
:- Mix by pipetting 10X
:- Spin down the plate at 2,000rpm for 1min
:- 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
'''''<span style="color:crimson"><u>Prep</u></span>'''''<br>
- Mix 600:1 ratio of Binding Buffer and 10ng/ul tRNA<br>
- For 24 rxn, I mixed 14.7mL of Binding Buffer with 24.5 ul of 10ng/ul tRNA
: - Add 601ul of Binding Buffer/tRNA mix to the column
:- Bind DNA to column by transfer bisulfite-treated DNA to the column and mixing by pipetting up and down for 5X. I rinse the well with small amount of Binding Buffer to transfer DNA to the column as much as possible
:- Spin down 14,000rpm for 30sec. <u>Discard spnt</u>
:- Wash with 100ul Wash buffer
:- Spin down 14,000rpm for 30sec
:- ''Incubate with 200ul of Desulphonation Buffer for 15min''
:- Spin down 14,000rpm for 30sec
:- Wash column with 200ul Wash Buffer.
:- Spin down 14,000rpm for 30sec. <u>Discard spnt</u>
:- Wash the column with 200ul Wash Buffer.
:- Spin down 14,000rpm for 2min
:- Elute converted DNA with warm (~60C) 31ul Elution Buffer. This should have ~30ul DNA left for PCR
=== 6) PCR amplification ===
:'''Primer info.'''
:- '''TruS_F:'''     5'- AATGATACGGCGACCACCGAGATC -3' (Tm = 67.1 C)
:- '''TruS_R:'''     5'- CAAGCAGAAGACGGCATACGAGAT -3'(Tm = 65.5 C)
:- I designed PCR primers a little longer (~3bp) than the ones used in Boyle P et al, 2012
==== <u>1st round PCR</u> ====
'''''<span style="color:crimson"><u>Prep</u></span>'''''<br>
:{| {{table}} class = wikitable
| align="center" style="background:#f0f0f0;"|'''Components'''
| align="center" style="background:#f0f0f0;"|'''Conc'''
| align="center" style="background:#f0f0f0;"|'''unit'''
| align="center" style="background:#f0f0f0;"|'''Final conc.'''
| align="center" style="background:#f0f0f0;"|'''unit'''
| align="center" style="background:#f0f0f0;"|'''Volume (ul)'''
| align="center" style="background:#f0f0f0;"|'''26 rxn mix'''
|-
| Bis-cvt DNA||||||||||30.00||0.00
|-
| 10X Reaction buffer||10||X||1||X||5.00||130.00
|-
| dNTP mix||10||mM||0.2||mM||1.00||26.00
|-
| TruS_F||10||uM||0.3||uM||1.50||39.00
|-
| TruS_R||10||uM||0.3||uM||1.50||39.00
|-
| PfuTurbo Cx||2.5||Unit/ul||1||unit||0.40||10.40
|-
| 50X SYBG||50||X||0.8||X||0.80||20.80
|-
| H2O||||||||||9.80||254.80
|}
:- Aliquot 20ul, add 30ul of bisulfite-treat adaptor-ligated DNA
::''95C for 2min --> [95C for 20sec -> '''60C for 30sec''' -> 72C for 1min] X 25 cycles --> 72C for 2min''<br>
:- Purified the 1st round amplicons with AMPurebeads 2X (1:1 ratio)


==== <u>AMPure bead purification</u> ====
'''''<span style="color:crimson"><u>Prep</u></span>'''''<br>
- Freshly prepare 20mL of 75% EtOH by mixing 15mL of 100% EtOH with 5mL of H2O<br>
- Add ~2.5mL of resuspened AMPure bead in 30mL reservoir
:- Add 50ul AMPure bead. Mix by pipetting 10x
:- Sit for 8min
:- Transfer to sit on magnet for 5min
:- Wash twice with 160ul freshly prepared 80% EtOH
:- Dry the bead for 3-5min
:- Resuspend with 50 H2O
:- Add 50ul of fresh AMPure bead. Mix by pipetting 10x
:- Sit for 5min
:- Transfer to sit on magnet for 5min
:- Wash twice with 160ul freshly prepared 80% EtOH
:- Dry the bead for 3-5min <u>(make sure that the beads are completely dried out to avoid EtOH inhibiting PCR)</u>
:- Resuspend the bead with 40ul H2O
:- Transfer purified 1st round amplicons to 8-tube strip with cap
:- Sit the strip tube on magnet before adding to the 2nd round PCR to avoid bead contamination in PCR
::- 32ul of bead purified 1st round amplicons will be added to the 2nd round PCR (32/40 -> 80%)
::- I saved the rest of bead purified 1st round amplicons
==== <u>2nd round PCR</u> ====
:{| {{table}} class = wikitable
| align="center" style="background:#f0f0f0;"|'''Components'''
| align="center" style="background:#f0f0f0;"|'''Conc'''
| align="center" style="background:#f0f0f0;"|'''unit'''
| align="center" style="background:#f0f0f0;"|'''Final conc.'''
| align="center" style="background:#f0f0f0;"|'''unit'''
| align="center" style="background:#f0f0f0;"|'''Volume (ul)'''
| align="center" style="background:#f0f0f0;"|'''26 rxn mix'''
|-
| Purified 1st round DNA||||||||||32.00||0.00
|-
| 5X Phusion HF buffer||5||X||1||X||10.00||260.00
|-
| dNTP mix||10||mM||0.2||mM||1.00||26.00
|-
| TruS_F||10||uM||0.3||uM||1.50||39.00
|-
| TruS_R||10||uM||0.3||uM||1.50||39.00
|-
| 50X SYBR||50||X||0.8||X||0.80||20.80
|-
| Phusion HF||2||unit/ul||1||unit||0.50||13.00
|-
| H2O||||||||||2.70||70.20
|}
:- Aliquot 18ul, add 32ul of bead purified 1st round amplicons<br>
::''98C for 2min --> [98C for 10sec -> 60C for 30sec -> 72C for 1min] X 22 cycles --> 72C for 2min''<br>
:- I did qPCR but let the reaction ran to 22 cycles for all samples.


== Results ==
== Results ==
: - Purified 40ul of 2nd round PCR with 1:1 ratio
:- Purified 40ul of 2nd round PCR with 1:1 ratio
: - Eluted with 40ul EB buffer
:- Eluted with 40ul EB buffer
: - Loaded 4ul of bead purified PCR products and also load 4ul some of unpurified PCR products
:- Loaded 4ul of bead purified PCR products and also load 4ul some of unpurified PCR products
[[File:ZhangLab_2 2014-04-23 13hr 46min_Apr#1_1-8bead_no1-no10-nobead.jpg| 400px]]
'''Ligation condition''':
: - <span style="color:blue">1ul of 1:20 diluted Illumina barcoded adaptors</span>
: - <span style="color:red">16C for 30min -> 4C for '''<u>14.5h</u>'''</span>
 
[[File:abc.png| 450px]] [[File:fgh_e.png| 439px]]
   
   
[[File:ZhangLab_2 2014-04-23 13hr 46min_Apr#1_9-16_bead_no11-no13-nobead.jpg| 400px]]
[[File:cde.png| 450px]]<br>
- Since I can not put all samples in the same gel, I stained the gels for 4min then replaced staining solution with 0.5X TBE buffer. I also used the same set up for all images
[[File:ZhangLab_2 2014-04-23 13hr 46min_Apr#1_17-24_bead_no17-no18-nobead.jpg| 400px]]
 
=== Discussions ===
== Discussions ==

Latest revision as of 10:26, 27 April 2014

scRRBS experiment: Apr#1[edit]

Link to calendar: [[1]]

  • I stopped doing scRRBS experiment for a while since I never get it works for single nuclei, and I think I should use the same barcoded methylated adaptors as used by Dr. Tang's group.
  • I found an old TrueSeq DNA Sample Prep kit in (Set A, part # 15013178) -20C. I then wanted to try these barcoded adaptors for ssRRBS while waiting for TrueSeq kit requested by Dr. Zhang. In this kit, I found only 6 indexed adaptors, including Ind2, Ind 4, Ind 5, Ind 6, Ind7 and Ind 12.
  • I have contacted with Dr. Tang's group for more information of scRRBS protocol in very details, and found there are many things I did differently from them. To be able to repeat the protocol and get it works for single cell, I followed everything noted by them.

More information from Dr. Tang's group[edit]

  • They used 80% of bead purified (two-round purification) 1st round amplicons as the template for the 2nd round PCR.
  • In both the 1st round and the 2nd round PCR, they did not do qPCR to monitor amplicons. They said that that the bisulfite-treated DNA was extremely low, so they did not monitor it.
- I think this might be the key reason that I never get success in single-nuclei experiment as I alway stopped reaction during the 2nd round PCR earlier before reaching 22 cycles (between 12-15 based on qPCR curve)
  • They heat-inactivate after MspI digestion, end-repair/dA-tailing, and methylated adaptors ligation
- MspI (not specify)
- End-repair/dA-tailing (I did 75C, 20min
- Ligation (65C, 20min) --> from Gu et al, 2011 (Nature Protocol). They suggest not to heat the lid since it could potentially destroy T4 DNA ligase.
  • They incubate ligation reaction at 16C, 30min --> 4C (at least 8hr). I previously incubate at 16C for at least 12h.
  • For PCR, thy used 200uM of dNTP and 300nM of primer-pair
  • I share all of these info with Yun Liu, the postdoctoral fellow in Dr. Feinberg's group, since he wants to try this protocol too.

NOTE[edit]

  • I used flow-sorted nuclei on 2014-04-10 for this experiment.
  • I still skipped unmethylated lambda DNA since I want to avoid the background in trial experiment. Once the protocol is settle, I will include it to see bisulfite conversion rate
  • I increased primer concentration in both 1st round and 2nd round PCR to 0.3mM or 300uM


Experimental Procedures[edit]

Plate layout[edit]

' 1 2 3
A 1 100 0
B 1 100 0
C 1 20 0
D 1 20 0
E 1 10 0
F 1 10 0
G 1 1 0
H 1 1 0
  • I include many of 0 nuclei since I want to see the consistency in all of them.
  • Some steps were not the same as Dr. Tang'g group suggestion since I got his email after I started experiment.

1) Cell lysis[edit]

Prep
- Thaw nuclei from -80C & spin down at 2,000rpm for 5min (96-well plate rotor, 5min)

- Add 1ul of protease
- Spin down the plate at 2,000rpm for 2min
- Mix by gentle pulse-vortexting for 10x
- Spin down the plate at 2,000rpm for 3min
- I tried to avoid pipetting up and down to mix the reaction to prevent nuclei/DNA lost.
- I notice solution was mixed well and no foaming generated
- I spin down the plate before and after mixing quite long to make sure that all reagents were collected to the bottom of the well
- Incubate at 50C for 3hr
- Heat inactivate at 75C for 30min
- Set program to hold at 15C
- Spin down the plate at 2,000rpm for 1min before continuing to next step


2) DNA fragmentation with MspI[edit]

  • Incubated released naked DNA with 9units of MspI in 18ul reaction at 37C for 3hr.

Prep
- Prepare MspI reaction mix

Components Volume (ul) 26x rxn mix
Lysed nuclei 5.00 0.00
10X Tango buffer 2.00 52.00
MspI 0.90 23.40
H2O 10.10 262.60
Total 18.00 338.00

- Aliquot 42ul of MspI enzyme mix to each tube in 8-tube strip

- Add 13ul to each well with multi-channel pipette
- Spin down the plate at 2,000rpm for 2min
- Mix by gentle pulse-vortexting for 10x
- Spin down the plate at 2,000rpm for 3min
- Incubate at 37C for 3hr
- Set program to hold at 15C
- Spin down the plate at 2,000rpm for 1min before continuing to next step


3) Gap-filling/dA-tailing[edit]

  • Add 5 units of Klenow fragment exo-, supplemented with 1mM dATP, 0.1 mM dGTP and 0.1 mM of dCTP in 20ul reaction. (Skip dTTP because enzyme cleaves C^CGG)

Prep
- Aliquot 5ul of dA:dC:dG mix (20mM:2mM:2mM) to each tube of 8-tube strip
- Aliquot 3.5ul of Klenow fragment exo- to each tube of 8-tube strip

- Add 1ul of dA:dC:dG solution mix to each well with multichannel pipette
- Add 1ul of Klenow fragment exo- to each well with multichannel pipette
- Add 13ul to each well with multi-channel pipette
- Spin down the plate at 2,000rpm for 2min
- Mix by gentle pulse-vortexting for 10x
- Spin down the plate at 2,000rpm for 3min
- I prefer to add reagents with multichannel pipette since I want to make sure no samples were missing during adding reagents and save a lot of my time. Indeed, less pipetting can avoid contamination.
- Incubate at 30C for 20min (for gap-filling) --> 37C for 20min (for extra dA-tailing)
- Heat inactivate enzyme at 75C for 10min
- Set program to hold at 4C
- Spin down the plate at 2,000rpm for 1min before continuing to next step


4) Methylated adaptor ligation[edit]

  • Ligate A-tailed DNA with 1ul of 1:20 diluted Illumina indexed methylated adaptor in total reaction 25ul at 16C for 30min and 4C for at least 8h (I did 14.5h --> next time will fix incubation time at 4C for consistency).

Index list
- I have only 6 indexes, I put Ind_2 and Ind_4 twice in 8-tube strip

Tube_1 Tube_2 Tube_3 Tube_4 Tube_5 Tube_6 Tube_7 Tube_8
Ind_2 Ind_4 Ind_5 Ind_6 Ind_7 Ind_12 Ind_2 Ind_4

Prep
- Mix 1ul of indexed methylated adaptors with 20ul H2O (on ice box)
- Prepare ligation reaction mix

Components Volume (ul) 28x rxn mix
dA-tailed reaction 20.00 0.00
10X Tango buffer 0.50 14.00
HC T4 DNA ligase (30units/ul) 1.00 28.00
10mM ATP 1.25 35.00
H2O 1.25 35.00
Total 24.00 112.00

- Aliquot 14ul of Ligation mix to each tube in 8-tube strip

- Add 1ul of diluted methylated adapter
- Add 4ul of ligation reaction mix
- Spin down the plate at 2000rpm for 2min
- Mix by gentle pulse-vortexting for 10x
- Spin down the plate at 2,000rpm for 3min
- Incubate at 16C for 30min -> 4C for 14.5h (no heat lid)
- Heat inactivate at 65C for 20min
- Spin down the plate at 2,000rpm for 1min before continuing to next step
Well ID Index Sample # Well ID Index Sample # Well ID Index Sample #
A1 Ind_2 #1 A2 Ind_2 #9 A3 Ind_2 #17
B1 Ind_4 #2 B2 Ind_4 #10 B3 Ind_4 #18
C1 Ind_5 #3 C2 Ind_5 #11 C3 Ind_5 #19
D1 Ind_6 #4 D2 Ind_6 #12 D3 Ind_6 #20
E1 Ind_7 #5 E2 Ind_7 #13 E3 Ind_7 #21
F1 Ind_12 #6 F2 Ind_12 #14 F3 Ind_12 #22
G1 Ind_2 #7 G2 Ind_2 #15 G3 Ind_2 #23
H1 Ind_4 #8 H2 Ind_4 #16 H3 Ind_4 #24

2014-04-22

5) Bisulfite conversion[edit]

  • I performed bisulfite conversion using the same procedure following manufacturer's instruction and elute with 31ul elution buffer.

Prep
- Prepare CT Conversion Reagent, by adding 850ul H2O, 50ul of Resuspension Buffer, and 300ul of Dilution Buffer to CT Conversion Reagent (for 25ul DNA sample --> reduce H2O from 900 to 850)

- Add125ul of complete CT Conversion Reagent to adaptor ligated DNA (no sample transfer to the new tube)
- Mix by pipetting 10X
- Spin down the plate at 2,000rpm for 1min
- 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

Prep
- Mix 600:1 ratio of Binding Buffer and 10ng/ul tRNA
- For 24 rxn, I mixed 14.7mL of Binding Buffer with 24.5 ul of 10ng/ul tRNA

- Add 601ul of Binding Buffer/tRNA mix to the column
- Bind DNA to column by transfer bisulfite-treated DNA to the column and mixing by pipetting up and down for 5X. I rinse the well with small amount of Binding Buffer to transfer DNA to the column as much as possible
- Spin down 14,000rpm for 30sec. Discard spnt
- Wash with 100ul Wash buffer
- Spin down 14,000rpm for 30sec
- Incubate with 200ul of Desulphonation Buffer for 15min
- Spin down 14,000rpm for 30sec
- Wash column with 200ul Wash Buffer.
- Spin down 14,000rpm for 30sec. Discard spnt
- Wash the column with 200ul Wash Buffer.
- Spin down 14,000rpm for 2min
- Elute converted DNA with warm (~60C) 31ul Elution Buffer. This should have ~30ul DNA left for PCR

6) PCR amplification[edit]

Primer info.
- TruS_F: 5'- AATGATACGGCGACCACCGAGATC -3' (Tm = 67.1 C)
- TruS_R: 5'- CAAGCAGAAGACGGCATACGAGAT -3'(Tm = 65.5 C)
- I designed PCR primers a little longer (~3bp) than the ones used in Boyle P et al, 2012

1st round PCR[edit]

Prep

Components Conc unit Final conc. unit Volume (ul) 26 rxn mix
Bis-cvt DNA 30.00 0.00
10X Reaction buffer 10 X 1 X 5.00 130.00
dNTP mix 10 mM 0.2 mM 1.00 26.00
TruS_F 10 uM 0.3 uM 1.50 39.00
TruS_R 10 uM 0.3 uM 1.50 39.00
PfuTurbo Cx 2.5 Unit/ul 1 unit 0.40 10.40
50X SYBG 50 X 0.8 X 0.80 20.80
H2O 9.80 254.80
- Aliquot 20ul, add 30ul of bisulfite-treat adaptor-ligated DNA
95C for 2min --> [95C for 20sec -> 60C for 30sec -> 72C for 1min] X 25 cycles --> 72C for 2min
- Purified the 1st round amplicons with AMPurebeads 2X (1:1 ratio)

AMPure bead purification[edit]

Prep
- Freshly prepare 20mL of 75% EtOH by mixing 15mL of 100% EtOH with 5mL of H2O
- Add ~2.5mL of resuspened AMPure bead in 30mL reservoir

- Add 50ul AMPure bead. Mix by pipetting 10x
- Sit for 8min
- Transfer to sit on magnet for 5min
- Wash twice with 160ul freshly prepared 80% EtOH
- Dry the bead for 3-5min
- Resuspend with 50 H2O
- Add 50ul of fresh AMPure bead. Mix by pipetting 10x
- Sit for 5min
- Transfer to sit on magnet for 5min
- Wash twice with 160ul freshly prepared 80% EtOH
- Dry the bead for 3-5min (make sure that the beads are completely dried out to avoid EtOH inhibiting PCR)
- Resuspend the bead with 40ul H2O
- Transfer purified 1st round amplicons to 8-tube strip with cap
- Sit the strip tube on magnet before adding to the 2nd round PCR to avoid bead contamination in PCR
- 32ul of bead purified 1st round amplicons will be added to the 2nd round PCR (32/40 -> 80%)
- I saved the rest of bead purified 1st round amplicons

2nd round PCR[edit]

Components Conc unit Final conc. unit Volume (ul) 26 rxn mix
Purified 1st round DNA 32.00 0.00
5X Phusion HF buffer 5 X 1 X 10.00 260.00
dNTP mix 10 mM 0.2 mM 1.00 26.00
TruS_F 10 uM 0.3 uM 1.50 39.00
TruS_R 10 uM 0.3 uM 1.50 39.00
50X SYBR 50 X 0.8 X 0.80 20.80
Phusion HF 2 unit/ul 1 unit 0.50 13.00
H2O 2.70 70.20
- Aliquot 18ul, add 32ul of bead purified 1st round amplicons
98C for 2min --> [98C for 10sec -> 60C for 30sec -> 72C for 1min] X 22 cycles --> 72C for 2min
- I did qPCR but let the reaction ran to 22 cycles for all samples.

Results[edit]

- Purified 40ul of 2nd round PCR with 1:1 ratio
- Eluted with 40ul EB buffer
- Loaded 4ul of bead purified PCR products and also load 4ul some of unpurified PCR products

Ligation condition:

- 1ul of 1:20 diluted Illumina barcoded adaptors
- 16C for 30min -> 4C for 14.5h

File:Abc.png File:Fgh e.png

File:Cde.png
- Since I can not put all samples in the same gel, I stained the gels for 4min then replaced staining solution with 0.5X TBE buffer. I also used the same set up for all images

Discussions[edit]