Noi/NOTES/2014-6-28: Difference between revisions

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[[noi:DMR220k_LabNotes#2014| '''Calendar''']]
[[noi:DMR220k_LabNotes#2014| '''Calendar''']]
==== Sample info ====
==== Sample info ====
* [[noi:Samples_from_Moores_Cancer_Center#2014-06-27| Solid tumor tissue from colon cancer patients (006-T)]]
* [[noi:Samples_from_Moores_Cancer_Center#2014-06-27| Solid tumor tissue from lung cancer patients (007-T)]]
{| {{table}} class = wikitable
{| {{table}} class = wikitable
| align="center" style="width:120px;background:#f0f0f0;"|'''Project ID'''
| align="center" style="width:120px;background:#f0f0f0;"|'''Project ID'''
Line 7: Line 9:
| align="center" style="width:80px;background:#f0f0f0;"|'''Conc. (ng/ul)'''
| align="center" style="width:80px;background:#f0f0f0;"|'''Conc. (ng/ul)'''
|-
|-
| UCSD-006-1||6-T_1||43.40
| UCSD-006-1||6-T-1||43.40
|-
|-
| UCSD-006-2||6-T_2||31.60
| UCSD-006-2||6-T-2||31.60
|-
|-
| UCSD-006-3||6-T_3||31.50
| UCSD-006-3||6-T-3||31.50
|-
|-
| UCSD-006-4||6-T_4||41.70
| UCSD-006-4||6-T-4||41.70
|-
|-
| UCSD-006-5||6-T_5||30.50
| UCSD-006-5||6-T-5||30.50
|-
|-
| UCSD-007-1||7-T_1||30.70
| UCSD-007-1||7-T-1||30.70
|-
|-
| UCSD-007-2||7-T_2||44.60
| UCSD-007-2||7-T-2||44.60
|-
|-
| UCSD-007-3||7-T_3||35.90
| UCSD-007-3||7-T-3||35.90
|-
|-
| UCSD-007-4||7-T_4||33.80
| UCSD-007-4||7-T-4||33.80
|-
|-
| UCSD-007-5||7-T_5||20.40
| UCSD-007-5||7-T-5||20.40
|}
|}
* X-Y_Z:  
* X-Y_Z:  
Line 31: Line 33:
** Y = sample type. T = tissue, P = plasma, S = serum
** Y = sample type. T = tissue, P = plasma, S = serum
** Z = sample ID
** Z = sample ID
== 1) MspI digestion ==
== 1) MspI digestion ==
* Conditions: Digest 100ng of DNA with 20U of MspI in 30ul reaction at 37C O/N (normally 3h) and heat inactivate at 65C 20min
* Conditions: Digest 100ng of DNA with 20U of MspI in 30ul reaction at 37C O/N (normally 3h) and heat inactivate at 65C 20min
Line 50: Line 53:
| align="center" style="width:80px;background:#f0f0f0;"|'''Code'''
| align="center" style="width:80px;background:#f0f0f0;"|'''Code'''
|-
|-
| 1||6-T_1.1||43.40||2.30||7.70||20.00||30.00||6T_1.1
| 1||6-T-1.1||43.40||2.30||7.70||20.00||30.00||6T-1.1
|-
|-
| 2||6-T_1.2||43.40||2.30||7.70||20.00||30.00||6T_1.2
| 2||6-T-1.2||43.40||2.30||7.70||20.00||30.00||6T-1.2
|-
|-
| 3||6-T_2.1||31.60||3.16||6.84||20.00||30.00||6T_2.1
| 3||6-T-2.1||31.60||3.16||6.84||20.00||30.00||6T-2.1
|-
|-
| 4||6-T_2.2||31.60||3.16||6.84||20.00||30.00||6T_2.2
| 4||6-T-2.2||31.60||3.16||6.84||20.00||30.00||6T-2.2
|-
|-
| 5||6-T_3.1||31.50||3.17||6.83||20.00||30.00||6T_3.1
| 5||6-T-3.1||31.50||3.17||6.83||20.00||30.00||6T-3.1
|-
|-
| 6||6-T_3.2||31.50||3.17||6.83||20.00||30.00||6T_3.2
| 6||6-T-3.2||31.50||3.17||6.83||20.00||30.00||6T-3.2
|-
|-
| 7||6-T_4.1||41.70||2.40||7.60||20.00||30.00||6T_4.1
| 7||6-T-4.1||41.70||2.40||7.60||20.00||30.00||6T-4.1
|-
|-
| 8||6-T_4.2||41.70||2.40||7.60||20.00||30.00||6T_4.2
| 8||6-T-4.2||41.70||2.40||7.60||20.00||30.00||6T-4.2
|-
|-
| 9||6-T_5.1||30.50||3.28||6.72||20.00||30.00||6T_5.1
| 9||6-T-5.1||30.50||3.28||6.72||20.00||30.00||6T-5.1
|-
|-
| 10||6-T_5.2||30.50||3.28||6.72||20.00||30.00||6T_5.2
| 10||6-T-5.2||30.50||3.28||6.72||20.00||30.00||6T-5.2
|-
|-
| 11||6-T_NTC||0.00||0.00||10.00||20.00||30.00||6T_NTC
| 11||6-T-NTC||0.00||0.00||10.00||20.00||30.00||6T-NTC
|}
|}
1.2) Lung sample, UCSD-007<br>
1.2) Lung sample, UCSD-007<br>
Line 83: Line 86:
| align="center" style="width:80px;background:#f0f0f0;"|'''Code'''
| align="center" style="width:80px;background:#f0f0f0;"|'''Code'''
|-
|-
| 1||7-T_1.1||30.70||3.26||6.74||20.00||30.00||7T_1.1
| 1||7-T-1.1||30.70||3.26||6.74||20.00||30.00||7T-1.1
|-
|-
| 2||7-T_1.2||30.70||3.26||6.74||20.00||30.00||7T_1.2
| 2||7-T-1.2||30.70||3.26||6.74||20.00||30.00||7T-1.2
|-
|-
| 3||7-T_2.1||44.60||2.24||7.76||20.00||30.00||7T_2.1
| 3||7-T-2.1||44.60||2.24||7.76||20.00||30.00||7T-2.1
|-
|-
| 4||7-T_2.2||44.60||2.24||7.76||20.00||30.00||7T_2.2
| 4||7-T-2.2||44.60||2.24||7.76||20.00||30.00||7T-2.2
|-
|-
| 5||7-T_3.1||35.90||2.79||7.21||20.00||30.00||7T_3.1
| 5||7-T-3.1||35.90||2.79||7.21||20.00||30.00||7T-3.1
|-
|-
| 6||7-T_3.2||35.90||2.79||7.21||20.00||30.00||7T_3.2
| 6||7-T-3.2||35.90||2.79||7.21||20.00||30.00||7T-3.2
|-
|-
| 7||7-T_4.1||33.80||2.96||7.04||20.00||30.00||7T_4.1
| 7||7-T-4.1||33.80||2.96||7.04||20.00||30.00||7T-4.1
|-
|-
| 8||7-T_4.2||33.80||2.96||7.04||20.00||30.00||7T_4.2
| 8||7-T-4.2||33.80||2.96||7.04||20.00||30.00||7T-4.2
|-
|-
| 9||7-T_5.1||20.40||4.90||5.10||20.00||30.00||7T_5.1
| 9||7-T-5.1||20.40||4.90||5.10||20.00||30.00||7T-5.1
|-
|-
| 10||7-T_5.2||20.40||4.90||5.10||20.00||30.00||7T_5.2
| 10||7-T-5.2||20.40||4.90||5.10||20.00||30.00||7T-5.2
|-
|-
| 11||7-T_NTC||0.00||0.00||10.00||20.00||30.00||7T_NTC
| 11||7-T-NTC||0.00||0.00||10.00||20.00||30.00||7T-NTC
|}
|}
==== <u>Msp I Reaction Mix</u> ====
==== <u>Msp I Reaction Mix</u> ====
Line 173: Line 176:
| 10x Ligation Buffer||3.00||72.00
| 10x Ligation Buffer||3.00||72.00
|-
|-
| T4 DNA Ligase (6000U/ul)||1.00||24.00
| T4 DNA Ligase (30U/ul)||1.00||24.00
|-
|-
| TruSeq Adaptor DNA||1.00||24.00
| TruSeq Adaptor DNA||1.00||24.00
Line 190: Line 193:
| align="center" style="width:70px;background:magenta;"|'''TruSeq Index'''
| align="center" style="width:70px;background:magenta;"|'''TruSeq Index'''
|-
|-
| 6-T_1.1||1||7-T_1.1||13
| 6-T-1.1||1||7-T-1.1||13
|-
|-
| 6-T_1.2||2||7-T_1.2||14
| 6-T-1.2||2||7-T-1.2||14
|-
|-
| 6-T_2.1||23||7-T_2.1||15
| 6-T-2.1||23||7-T-2.1||15
|-
|-
| 6-T_2.2||4||7-T_2.2||16
| 6-T-2.2||4||7-T-2.2||16
|-
|-
| 6-T_3.1||5||7-T_3.1||27
| 6-T-3.1||5||7-T-3.1||27
|-
|-
| 6-T_3.2||6||7-T_3.2||18
| 6-T-3.2||6||7-T-3.2||18
|-
|-
| 6-T_4.1||7||7-T_4.1||19
| 6-T-4.1||7||7-T-4.1||19
|-
|-
| 6-T_4.2||8||7-T_4.2||20
| 6-T-4.2||8||7-T-4.2||20
|-
|-
| 6-T_5.1||25||7-T_5.1||21
| 6-T-5.1||25||7-T-5.1||21
|-
|-
| 6-T_5.2||10||7-T_5.2||22
| 6-T-5.2||10||7-T-5.2||22
|-
|-
| 6-T_NTC||10||7-T_NTC||22
| 6-T-NTC||10||7-T-NTC||22
|}
|}
:- Add 9ul of ligation reaction mix with multichannel pipette
:- Add 9ul of ligation reaction mix with multichannel pipette
Line 241: Line 244:
<br>
<br>
== Bisulfite conversion ==
== 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.
* Performed bisulfite conversion using LifeTech MethylCode™ Bisulfite Conversion Kit and did the same procedure following manufacturer's instruction and elute with 30ul Elution buffer.
'''''<span style="color:crimson"><u>Prep</u></span>'''''<br>
'''''<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
:- Prepare 2 tubes of complete CT Conversion Reagent by adding 800ul H2O, 50ul Resuspension Buffer, and 300ul Dilution Buffer
Line 274: Line 277:
:- Spin down column at 12,000 rpm for 1min
:- Spin down column at 12,000 rpm for 1min
:- Continue to amplification (During elution step, PCR mix should be prepared)
:- Continue to amplification (During elution step, PCR mix should be prepared)
== Amplification ==
== Amplification ==
=== TEST PCR ===
=== TEST PCR ===
Line 344: Line 348:
:: ''95C for 2min --> [95C for 20sec -> 60C for 30sec -> 72C for 1min] X 14 cycles --> 72C for 2min''
:: ''95C for 2min --> [95C for 20sec -> 60C for 30sec -> 72C for 1min] X 14 cycles --> 72C for 2min''
:- Stopped at 14X cycles based on TEST PCR
:- Stopped at 14X cycles based on TEST PCR
:- I pooled the two PCR replicates (total ~90ul for each samples) and purified with 1.2x volume AMPure beads and eluted with 55ul EB buffer
:- I pooled the two PCR replicates (total ~90ul for each samples) and purified with 1x volume AMPure beads and eluted with 55ul EB buffer
:- I did PAGE quantification by loading 3ul of bead-purified sequencing libraries
:- I did PAGE quantification by loading 3ul of bead-purified sequencing libraries
  [[File:ZhangLab_2 2014-07-02 19hr 34min_PQ_6T_100ngRRBS.jpg| 500px]]  
  [[File:ZhangLab_2 2014-07-02 19hr 34min_PQ_6T_100ngRRBS.jpg| 500px]]  
Line 361: Line 365:
| align="center" style="width:80px;background:#f0f0f0;"|'''Amount in 50ul (ng)'''
| align="center" style="width:80px;background:#f0f0f0;"|'''Amount in 50ul (ng)'''
|-
|-
| 6-T_1.1||1||6.53||326.73
| 6-T-1.1||1||6.53||326.73
|-
|-
| 6-T_1.2||2||9.91||495.56
| 6-T-1.2||2||9.91||495.56
|-
|-
| 6-T_2.1||23||2.00||100.20
| 6-T-2.1||23||2.00||100.20
|-
|-
| 6-T_2.2||4||7.14||356.87
| 6-T-2.2||4||7.14||356.87
|-
|-
| 6-T_3.1||5||10.60||530.01
| 6-T-3.1||5||10.60||530.01
|-
|-
| 6-T_3.2||6||6.70||334.87
| 6-T-3.2||6||6.70||334.87
|-
|-
| 6-T_4.1||7||6.19||309.49
| 6-T-4.1||7||6.19||309.49
|-
|-
| 6-T_4.2||8||0.71||35.55
| 6-T-4.2||8||0.71||35.55
|-
|-
| 6-T_5.1||25||4.31||215.60
| 6-T-5.1||25||4.31||215.60
|-
|-
| 6-T_5.2||10||8.26||412.83
| 6-T-5.2||10||8.26||412.83
|}
|}
<u> 007-T samples</u>
<u> 007-T samples</u>
* Value of sample 7-T-4.1 is the repeat sample (will be added)
* Value of sample 7-T-4.1 is the repeated sample (will be added)
{| {{table}} border = 1
{| {{table}} border = 1
| align="center" style="width:120px;background:#f0f0f0;"|'''Sample'''
| align="center" style="width:120px;background:#f0f0f0;"|'''Sample'''
Line 389: Line 393:
| align="center" style="width:80px;background:#f0f0f0;"|'''Amount in 50ul (ng)'''
| align="center" style="width:80px;background:#f0f0f0;"|'''Amount in 50ul (ng)'''
|-
|-
| 7-T_1.1||13||5.12||255.78
| 7-T-1.1||13||5.12||255.78
|-
| 7-T-1.2||14||3.65||182.39
|-
| 7-T-2.1||15||4.18||208.94
|-
| 7-T-2.2||16||6.55||327.39
|-
| 7-T-3.1||27||2.70||135.19
|-
| 7-T-3.2||18||3.72||185.97
|-
| <span style="color:crimson">7-T-4.1</span>||19||9.30||465.00
|-
| 7-T-4.2||20||3.59||179.72
|-
| 7-T-5.1||21||5.76||288.10
|-
| 7-T-5.2||22||3.82||190.84
|}<br>
==== <u>Library pooling</u> ====
=====<span style="color:mediumblue"><u> 006-T samples</u></span>=====
* Since I may need to sequence only one replicate, I combine sequencing libraries into two set; set 1 and set2. Set 1 set the first priority, so I included sample 6-T-2_2 in set one as sample 6-T-2_1had very faint signal. I included sample 6-T_2.1 in set 2
<span style="color:mediumblue">NP-RRBS-6T-set1_100ng-Jun28</span>
{| {{table}} border = 1
| align="center" style="width:120px;background:#f0f0f0;"|'''Sample'''
| align="center" style="width:60px;background:#f0f0f0;"|'''Index'''
| align="center" style="width:80px;background:#f0f0f0;"|'''Conc. (ng/ul)'''
| align="center" style="width:80px;background:#f0f0f0;"|'''Volume for 120ng (ul)'''
|-
| 6-T_1.1||1||6.53||18.36
|-
| <span style="color:crimson">6-T_2.2</span>||4||7.14||16.81
|-
| 6-T-3.1||5||10.60||11.32
|-
| 6-T-4.1||7||6.19||19.39
|-
| 6-T-5.1||25||4.31||27.83
|}
:- Add 20ul of 6X loading dye and load 37.9ul in 3 well of 5-well 6% TBE gel
<span style="color:mediumblue">NP-RRBS-6T-set2_100ng-Jun28</span>
* Set 2 I include 4 libraries and do PAGE size selection of sample 6-T-4.2 separately since it had very faint smear.
{| {{table}} border = 1
| align="center" style="width:120px;background:#f0f0f0;"|'''Sample'''
| align="center" style="width:60px;background:#f0f0f0;"|'''Index'''
| align="center" style="width:80px;background:#f0f0f0;"|'''Conc. (ng/ul)'''
| align="center" style="width:80px;background:#f0f0f0;"|'''Volume for 100ng (ul)'''
|-
| 6-T-1.2||2||9.91||10.09
|-
| 6-T-2.1||23||2.00||49.90
|-
| 6-T-3.2||6||6.70||14.93
|-
| 6-T-5.2||10||8.26||12.11
|}
:- Add 20ul of 6X loading dye and load 35.68 in 3 well of 5-well 6% TBE gel
<span style="color:mediumblue">NP-RRBS-6T-4_2-set2_100ng-Jun28</span>
{| {{table}} border = 1
| align="center" style="width:120px;background:#f0f0f0;"|'''Sample'''
| align="center" style="width:60px;background:#f0f0f0;"|'''Index'''
| align="center" style="width:80px;background:#f0f0f0;"|'''Conc. (ng/ul)'''
| align="center" style="width:80px;background:#f0f0f0;"|'''Volume (ul)'''
|-
| 6-T-4.2||8||0.71||50.00
|}
:- Add 10ul of 6X loading dye and load 60ul in 1 well of 5-well 6% TBE gel
 
=====<span style="color:red"><u> 007-T samples</u></span>=====
<span style="color:red">NP-RRBS-7T-set1_100ng-Jun28</span>
{| {{table}} border = 1
| align="center" style="width:120px;background:#f0f0f0;"|'''Sample'''
| align="center" style="width:60px;background:#f0f0f0;"|'''Index'''
| align="center" style="width:80px;background:#f0f0f0;"|'''Conc. (ng/ul)'''
| align="center" style="width:80px;background:#f0f0f0;"|'''Volume for 100ng (ul)'''
|-
| 7-T-1.1||13||5.12||19.55
|-
| 7-T-2.1||15||4.18||23.93
|-
| 7-T-3.1||27||2.70||36.98
|-
| 7-T-4.1||19||9.30||10.75
|-
| 7-T-5.1||21||5.76||17.35
|}
:- Add 25ul of 6X loading dye and load 44.52ul in 3 well of 5-well 6% TBE gel
<span style="color:red">NP-RRBS-7T-set2_100ng-Jun28</span>
{| {{table}} border = 1
| align="center" style="width:120px;background:#f0f0f0;"|'''Sample'''
| align="center" style="width:60px;background:#f0f0f0;"|'''Index'''
| align="center" style="width:80px;background:#f0f0f0;"|'''Conc. (ng/ul)'''
| align="center" style="width:80px;background:#f0f0f0;"|'''Volume for 100ng (ul)'''
|-
| 7-T-1.2||14||3.65||27.41
|-
|-
| 7-T_1.2||14||3.65||182.39
| 7-T-2.2||16||6.55||15.27
|-
|-
| 7-T_2.1||15||4.18||208.94
| 7-T-3.2||18||3.72||26.89
|-
|-
| 7-T_2.2||16||6.55||327.39
| 7-T-4.2||20||3.59||27.82
|-
|-
| 7-T_3.1||27||2.70||135.19
| 7-T-5.2||22||3.82||26.20
|}
:- Add 30ul of 6X loading dye and load 51.2ul in 3 well of 5-well 6% TBE gel<br>
==== PAGE size selection ====
[[File:ZhangLab_2 2014-07-06 17hr 56min_gel1.jpg|380px]]
- NP-RRBS-6T_set1_100ng-Jun28: Resuspend with 12ul H2O x 3tubes --> volume 36ul, 4ul for PAGE verification
 
[[File:ZhangLab_2 2014-07-06 17hr 58min_gel2.jpg|380px]]
- NP-RRBS-6T_set2_100ng-Jun28: Resuspend with 12ul H2O x 3tubes --> volume 36ul, 4ul for PAGE verification
- NP-RRBS-6T_4_2_set2_100ng-Jun28: Resuspend with 12ul H2O x 1tube--> volume 12ul, no PAGE verification
[[File:ZhangLab_2 2014-07-06 18hr 03min_gel3.jpg|380px]]
  - NP-RRBS-7T_set1_100ng-Jun28: Resuspend with 12ul H2O x 3tubes --> volume 36ul, 4ul for PAGE verification
 
[[File:ZhangLab_2 2014-07-06 18hr 04min_gel4.jpg|380px]]
- NP-RRBS-7T_set2_100ng-Jun28: Resuspend with 12ul H2O x 3tubes --> volume 36ul, 4ul for PAGE verification
 
==== PAGE verification ====
 
 
 
----
 
=== 2014-07-08 ===
==== Preparing sequencing library pool for IGM ====
* We will do HiSeq Rapid run, PE 100+7bp
* Spike in with 5% PhiX in each lane
:1). '''NP_RRBS_6T_100ng_20140708''' (Loaded in lane 1)
:{| {{table}} class = wikitable
| align="center" style="width:220px;background:#f0f0f0;"|'''Seq. Library'''
| align="center" style="width:60px;background:#f0f0f0;"|'''# of ind. Library'''
| align="center" style="width:80px;background:#f0f0f0;"|'''# of read for each (M)'''
| align="center" style="width:80px;background:#f0f0f0;"|'''Total # of reads (M)'''
| align="center" style="width:60px;background:#f0f0f0;"|'''Loading %'''
| align="center" style="width:80px;background:#f0f0f0;"|'''Conc. (nM)'''
| align="center" style="width:80px;background:#f0f0f0;"|'''Ratio of % to 6T-set1'''
| align="center" style="width:80px;background:#f0f0f0;"|'''# of mole to pool (nmole)'''
| align="center" style="width:80px;background:#f0f0f0;"|'''Volume to pool (ul)'''
|-
|-
| 7-T_3.2||18||3.72||185.97
| NP-RRBS-6T_set1_100ng-Jun28||5||10||50||50||11.63||1||50||4.30
|-
|-
| 7-T_4.1||19||9.30||465.00
| NP-RRBS-6T_set2_100ng-Jun28||4||10||40||40||9.01||0.8||40||4.44
|-
|-
| 7-T_4.2||20||3.59||179.72
| NP-RRBS-6T_4_2_set2_100ng-Jun28||1||10||10||10||2.44||0.2||10||4.10
|-
|-
| 7-T_5.1||21||5.76||288.10
| ||||||||||||Total||100||12.84
|-
|-
| 7-T_5.2||22||3.82||190.84
| ||||||||||||Final conc.||7.79||nM
|}
|}


==== Library pool ====
:2). '''NP_RRBS_7T_100ng_20140708''' (Loaded in lane 2)
:{| {{table}} class = wikitable
| align="center" style="width:220px;background:#f0f0f0;"|'''Seq. Library'''
| align="center" style="width:60px;background:#f0f0f0;"|'''# of ind. Library'''
| align="center" style="width:80px;background:#f0f0f0;"|'''# of read for each (M)'''
| align="center" style="width:80px;background:#f0f0f0;"|'''Total # of reads (M)'''
| align="center" style="width:60px;background:#f0f0f0;"|'''Loading %'''
| align="center" style="width:80px;background:#f0f0f0;"|'''Conc. (nM)'''
| align="center" style="width:80px;background:#f0f0f0;"|'''Ratio of % to 7T-set1'''
| align="center" style="width:80px;background:#f0f0f0;"|'''# of mole to pool (nmole)'''
| align="center" style="width:80px;background:#f0f0f0;"|'''Volume to pool (ul)'''
|-
| NP-RRBS-7T_set1_100ng-Jun28||5||10||50||50||15.6||1||100||6.41
|-
| NP-RRBS-7T_set2_100ng-Jun28||5||10||50||50||13.47||1||100||7.42
|-
| ||||||||||||Total||200||13.83
|-
| ||||||||||||Final conc.||14.46||nM
|}

Latest revision as of 08:04, 18 December 2015

Preparation of RRBS sequencing libraries of solid tumor tissue sample from colon and lung cancer patients[edit]

Calendar

Sample info[edit]

Project ID Zhang lab ID Conc. (ng/ul)
UCSD-006-1 6-T-1 43.40
UCSD-006-2 6-T-2 31.60
UCSD-006-3 6-T-3 31.50
UCSD-006-4 6-T-4 41.70
UCSD-006-5 6-T-5 30.50
UCSD-007-1 7-T-1 30.70
UCSD-007-2 7-T-2 44.60
UCSD-007-3 7-T-3 35.90
UCSD-007-4 7-T-4 33.80
UCSD-007-5 7-T-5 20.40
  • 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[edit]

  • Conditions: Digest 100ng of DNA with 20U of MspI in 30ul reaction at 37C O/N (normally 3h) and heat inactivate at 65C 20min
  • For 100ng DNA input, I used 2ul (20U) of MspI.
  • I spike in 1% of unmeth lambda DNA (1ng)
  • I actually start MspI at late night of 2014-06-27, so I incubated it O/N as it doesn't hurt to incubate DNA with MspI longer than 3h.
  • I do two replicates for each sample


MspI digestion set up[edit]

1.1) Colon samples, UCSD-006

# Sample Conc. (ng/ul) Volume for 100ng (ul) H2O to 10ul MspI Mix Total Code
1 6-T-1.1 43.40 2.30 7.70 20.00 30.00 6T-1.1
2 6-T-1.2 43.40 2.30 7.70 20.00 30.00 6T-1.2
3 6-T-2.1 31.60 3.16 6.84 20.00 30.00 6T-2.1
4 6-T-2.2 31.60 3.16 6.84 20.00 30.00 6T-2.2
5 6-T-3.1 31.50 3.17 6.83 20.00 30.00 6T-3.1
6 6-T-3.2 31.50 3.17 6.83 20.00 30.00 6T-3.2
7 6-T-4.1 41.70 2.40 7.60 20.00 30.00 6T-4.1
8 6-T-4.2 41.70 2.40 7.60 20.00 30.00 6T-4.2
9 6-T-5.1 30.50 3.28 6.72 20.00 30.00 6T-5.1
10 6-T-5.2 30.50 3.28 6.72 20.00 30.00 6T-5.2
11 6-T-NTC 0.00 0.00 10.00 20.00 30.00 6T-NTC

1.2) Lung sample, UCSD-007

# Sample Conc. (ng/ul) Volume for 100ng (ul) H2O to 10ul MspI Mix Total Code
1 7-T-1.1 30.70 3.26 6.74 20.00 30.00 7T-1.1
2 7-T-1.2 30.70 3.26 6.74 20.00 30.00 7T-1.2
3 7-T-2.1 44.60 2.24 7.76 20.00 30.00 7T-2.1
4 7-T-2.2 44.60 2.24 7.76 20.00 30.00 7T-2.2
5 7-T-3.1 35.90 2.79 7.21 20.00 30.00 7T-3.1
6 7-T-3.2 35.90 2.79 7.21 20.00 30.00 7T-3.2
7 7-T-4.1 33.80 2.96 7.04 20.00 30.00 7T-4.1
8 7-T-4.2 33.80 2.96 7.04 20.00 30.00 7T-4.2
9 7-T-5.1 20.40 4.90 5.10 20.00 30.00 7T-5.1
10 7-T-5.2 20.40 4.90 5.10 20.00 30.00 7T-5.2
11 7-T-NTC 0.00 0.00 10.00 20.00 30.00 7T-NTC

Msp I Reaction Mix[edit]

Components 1x rxn 24 rxn Mix
MspI (10U/ul) 2.00 48.00
10x Tango Buffer 3.00 72.00
1ng/ul unmeth-lambda DNA 1.00 24.00
H2O 14.00 336.00
Total 20.00

Prep
- Aliquot MspI reaction mix to 8-tube strip. 65ul for tube #1-3, #6-8 and 44ul for tube #4-5

- Add H2O to each tube
- Add 100ng of DNA to each tube
- Add 20ul 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 (~13.5h) --> 65C, 20min --> hold at 4C (set heat lid)


2) End-repair/dA-tailing[edit]

Prep
- Mix 26ul of Klenow exo- with 26ul of dA:dC:dG (stock 20mM:2mM:2mM) just right before adding to MspI digested DNA
- Aliquot Klenow, exo- and dA:dC:dG mix to 8-tube strip. 7ul for tube #1-3, #6-8 and 5ul for tube #4-6

- 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)
  • 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[edit]

Prep
- Freshly prepare 8mL of 75% EtOH by mixing 6mL of 100% EtOH with 2mL of H2O (prepare when the tubes sit on magnet)
- Aliquot 1400ul of AMPure beads in 30mL reservoir and leave at RT

- Add 64ul AMPure bead with multichannel pipette. 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!)


3) Methylated adaptor ligation[edit]

  • 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

Ligation reaction set up[edit]

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[edit]

Components 1x rxn 24rxn Mix
dA-tailed DNA 20.00 0.00
10x Ligation Buffer 3.00 72.00
T4 DNA Ligase (30U/ul) 1.00 24.00
TruSeq Adaptor DNA 1.00 24.00
H2O 5.00 120.00
Total 30.00

Prep
- Aliquot ligation reaction mix to 8-tube strip. 29ul for tube #1-3, #6-8 and 20ul for tube #4-5

- Add 1ul of non-diluted TruSeq adaptors and pipette few times to make sure adaptors were added to the reaction
Sample TruSeq Index Sample TruSeq Index
6-T-1.1 1 7-T-1.1 13
6-T-1.2 2 7-T-1.2 14
6-T-2.1 23 7-T-2.1 15
6-T-2.2 4 7-T-2.2 16
6-T-3.1 5 7-T-3.1 27
6-T-3.2 6 7-T-3.2 18
6-T-4.1 7 7-T-4.1 19
6-T-4.2 8 7-T-4.2 20
6-T-5.1 25 7-T-5.1 21
6-T-5.2 10 7-T-5.2 22
6-T-NTC 10 7-T-NTC 22
- Add 9ul 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, 24h (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)
  • Before continue to bisulfite conversion, purify adaptor-ligated DNA with 2x volume diluted AMPure beads (60ul each) and resuspend the bead with 31ul EB buffer

2014-06-29[edit]

AMPure bead purification[edit]

Prep
- Freshly prepare 8mL of 75% EtOH by mixing 6mL of 100% EtOH with 2mL of H2O (prepare when the tubes sit on magnet)
- Aliquot 670ul of AMPure beads and 670ul of 20% PEG 8000/5M NaCl in 1.5mL tube, mix well and leave at RT (forgot to dilute, used beads directly)

- Add 60ul AMPure bead with multichannel pipette. 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.
- Wait for 2min.
- Transfer to sit on magnet for 2 min and transfer 30ul of adaptor-ligated DNA to new strip tubes for bisulfite conversion (discard the beads)
- Continue to bisulfite conversion


Bisulfite conversion[edit]

  • Performed bisulfite conversion using LifeTech MethylCode™ Bisulfite Conversion Kit and did the same procedure following manufacturer's instruction and elute with 30ul 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)

Amplification[edit]

TEST PCR[edit]

Components Conc unit Final conc./amount unit Volume (ul) 25 rxn mix
Bis-cvt DNA 6.00 0.00
10X Reaction buffer 10 X 1 X 2.00 50.00
dNTP mix 10 mM 0.25 mM 0.50 12.50
TruS_F/R 10 uM 0.3 uM 0.60 15.00
50X SYBG 50 X 0.8 X 0.32 8.00
PfuTurbo Cx 2.5 Unit/ul 1 unit 0.40 10.00
H2O 10.18 254.50
Total 20.00
- Aliquot 14 + 6ul DNA template
- Mix well. Split 2X of 8.5ul
95C for 2min --> [95C for 20sec -> 60C for 30sec -> 72C for 1min] X [14X & 16X] --> 72C for 2min
  • There was error to realplex during annealing step of 15 cycles. I needed to remove samples. I loaded only 14X to save the gel and plan to loaded 16 cycles PCR if the intensity of 14X is too low.
- Add 10ul of 2X loading dye to PCR product and load 10ul PCR product/dyemix in 12% gel and run at 250 volts for 40min
Colon tumor samples
File:ZhangLab 2 2014-06-30 17hr 16min 6T 100ngRRBS TEST-14X.jpg
 
Lung tumor samples
File:ZhangLab 2 2014-06-30 17hr 16min 7T 100ngRRBS TEST-14X-e.jpgg
  • I do not know why sample 7-T_3.2 and 7-T_4.1 failed. May need to check with Dr. Zhang if he want to sequence the two duplicates or only 1 library for each sample.
  • No need to load 16 cycles PCR since 14 cycles look good and not too faint or over-amplified.
  • I need to repeat 7-T_3.2 and 7-T_4.1. I plan to repeat both of them in two duplicates.
  • Continued to prepare sequencing library by doing PCR in 100ul reaction with 30% BIS-DNA.
Components Conc unit Final conc./amount unit Volume (ul) 20.5
Bis-cvt DNA 30.00 0.00
10X Reaction buffer 10 X 1 X 10.00 205.00
dNTP mix 10 mM 0.25 mM 2.50 51.25
TruS_F/R 10 uM 0.3 uM 3.00 61.50
50X SYBG 50 X 0.8 X 1.60 32.80
PfuTurbo Cx 2.5 Unit/ul 1 unit 2.00 41.00
H2O 50.90 1043.45
Total 100.00
- Aliquot 70 + 30ul DNA template
- Mix well. Split 2X of 50ul (actual ~48ul)
95C for 2min --> [95C for 20sec -> 60C for 30sec -> 72C for 1min] X 14 cycles --> 72C for 2min
- Stopped at 14X cycles based on TEST PCR
- I pooled the two PCR replicates (total ~90ul for each samples) and purified with 1x volume AMPure beads and eluted with 55ul EB buffer
- I did PAGE quantification by loading 3ul of bead-purified sequencing libraries
File:ZhangLab 2 2014-07-02 19hr 34min PQ 6T 100ngRRBS.jpg 

File:ZhangLab 2 2014-07-02 19hr 34min PQ 7T 100ngRRBS.jpg
  • It's so wired for sample 6T_4.2 since it looked fine in TEST PCR, but it's very faint after amplification in 50ul and bead purified.
Repeat smaple 7T_4.1 (also include 7T_4.2)
File:ZhangLab 2 2014-07-03 17hr 35min PQ repeat 7T 4.jpg
* I reloaded sample 6T_2.1 and 6T_4.2 again. To confirm that they had very low concentration.

PAGE quantification results[edit]

006-T samples

Sample Index Conc. (ng/ul) Amount in 50ul (ng)
6-T-1.1 1 6.53 326.73
6-T-1.2 2 9.91 495.56
6-T-2.1 23 2.00 100.20
6-T-2.2 4 7.14 356.87
6-T-3.1 5 10.60 530.01
6-T-3.2 6 6.70 334.87
6-T-4.1 7 6.19 309.49
6-T-4.2 8 0.71 35.55
6-T-5.1 25 4.31 215.60
6-T-5.2 10 8.26 412.83

007-T samples

  • Value of sample 7-T-4.1 is the repeated sample (will be added)
Sample Index Conc. (ng/ul) Amount in 50ul (ng)
7-T-1.1 13 5.12 255.78
7-T-1.2 14 3.65 182.39
7-T-2.1 15 4.18 208.94
7-T-2.2 16 6.55 327.39
7-T-3.1 27 2.70 135.19
7-T-3.2 18 3.72 185.97
7-T-4.1 19 9.30 465.00
7-T-4.2 20 3.59 179.72
7-T-5.1 21 5.76 288.10
7-T-5.2 22 3.82 190.84


Library pooling[edit]

006-T samples[edit]
  • Since I may need to sequence only one replicate, I combine sequencing libraries into two set; set 1 and set2. Set 1 set the first priority, so I included sample 6-T-2_2 in set one as sample 6-T-2_1had very faint signal. I included sample 6-T_2.1 in set 2

NP-RRBS-6T-set1_100ng-Jun28

Sample Index Conc. (ng/ul) Volume for 120ng (ul)
6-T_1.1 1 6.53 18.36
6-T_2.2 4 7.14 16.81
6-T-3.1 5 10.60 11.32
6-T-4.1 7 6.19 19.39
6-T-5.1 25 4.31 27.83
- Add 20ul of 6X loading dye and load 37.9ul in 3 well of 5-well 6% TBE gel

NP-RRBS-6T-set2_100ng-Jun28

  • Set 2 I include 4 libraries and do PAGE size selection of sample 6-T-4.2 separately since it had very faint smear.
Sample Index Conc. (ng/ul) Volume for 100ng (ul)
6-T-1.2 2 9.91 10.09
6-T-2.1 23 2.00 49.90
6-T-3.2 6 6.70 14.93
6-T-5.2 10 8.26 12.11
- Add 20ul of 6X loading dye and load 35.68 in 3 well of 5-well 6% TBE gel

NP-RRBS-6T-4_2-set2_100ng-Jun28

Sample Index Conc. (ng/ul) Volume (ul)
6-T-4.2 8 0.71 50.00
- Add 10ul of 6X loading dye and load 60ul in 1 well of 5-well 6% TBE gel
007-T samples[edit]

NP-RRBS-7T-set1_100ng-Jun28

Sample Index Conc. (ng/ul) Volume for 100ng (ul)
7-T-1.1 13 5.12 19.55
7-T-2.1 15 4.18 23.93
7-T-3.1 27 2.70 36.98
7-T-4.1 19 9.30 10.75
7-T-5.1 21 5.76 17.35
- Add 25ul of 6X loading dye and load 44.52ul in 3 well of 5-well 6% TBE gel

NP-RRBS-7T-set2_100ng-Jun28

Sample Index Conc. (ng/ul) Volume for 100ng (ul)
7-T-1.2 14 3.65 27.41
7-T-2.2 16 6.55 15.27
7-T-3.2 18 3.72 26.89
7-T-4.2 20 3.59 27.82
7-T-5.2 22 3.82 26.20
- Add 30ul of 6X loading dye and load 51.2ul in 3 well of 5-well 6% TBE gel

PAGE size selection[edit]

File:ZhangLab 2 2014-07-06 17hr 56min gel1.jpg
- NP-RRBS-6T_set1_100ng-Jun28: Resuspend with 12ul H2O x 3tubes --> volume 36ul, 4ul for PAGE verification
 
File:ZhangLab 2 2014-07-06 17hr 58min gel2.jpg
- NP-RRBS-6T_set2_100ng-Jun28: Resuspend with 12ul H2O x 3tubes --> volume 36ul, 4ul for PAGE verification
- NP-RRBS-6T_4_2_set2_100ng-Jun28: Resuspend with 12ul H2O x 1tube--> volume 12ul, no PAGE verification

File:ZhangLab 2 2014-07-06 18hr 03min gel3.jpg
 - NP-RRBS-7T_set1_100ng-Jun28: Resuspend with 12ul H2O x 3tubes --> volume 36ul, 4ul for PAGE verification
 
File:ZhangLab 2 2014-07-06 18hr 04min gel4.jpg
- NP-RRBS-7T_set2_100ng-Jun28: Resuspend with 12ul H2O x 3tubes --> volume 36ul, 4ul for PAGE verification

PAGE verification[edit]


2014-07-08[edit]

Preparing sequencing library pool for IGM[edit]

  • We will do HiSeq Rapid run, PE 100+7bp
  • Spike in with 5% PhiX in each lane
1). NP_RRBS_6T_100ng_20140708 (Loaded in lane 1)
Seq. Library # of ind. Library # of read for each (M) Total # of reads (M) Loading % Conc. (nM) Ratio of % to 6T-set1 # of mole to pool (nmole) Volume to pool (ul)
NP-RRBS-6T_set1_100ng-Jun28 5 10 50 50 11.63 1 50 4.30
NP-RRBS-6T_set2_100ng-Jun28 4 10 40 40 9.01 0.8 40 4.44
NP-RRBS-6T_4_2_set2_100ng-Jun28 1 10 10 10 2.44 0.2 10 4.10
Total 100 12.84
Final conc. 7.79 nM
2). NP_RRBS_7T_100ng_20140708 (Loaded in lane 2)
Seq. Library # of ind. Library # of read for each (M) Total # of reads (M) Loading % Conc. (nM) Ratio of % to 7T-set1 # of mole to pool (nmole) Volume to pool (ul)
NP-RRBS-7T_set1_100ng-Jun28 5 10 50 50 15.6 1 100 6.41
NP-RRBS-7T_set2_100ng-Jun28 5 10 50 50 13.47 1 100 7.42
Total 200 13.83
Final conc. 14.46 nM