Noi/NOTES/2014-6-2: Difference between revisions

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= RRBS library preparation of colon tumor tissue samples from Biochain =
= RRBS library preparation of colon tumor tissue samples from Biochain =
* [[http://genome-tech.ucsd.edu/LabNotes/index.php/noi:DMR220k_LabNotes '''Link to calendar''']]
* [[http://genome-tech.ucsd.edu/LabNotes/index.php/noi:DMR220k_LabNotes '''Link to calendar''']]
==== Sample list  ====
==== Sample list and details ====
* Sample type: genomic DNA from primary tumor tissue
* Sample type: genomic DNA from primary tumor tissue
** Colon tumor tissue, frozen (CTT-frozen): re-quantified concentration with Qubit dsDNA HS assay  
** Colon tumor tissue, frozen (CTT-frozen): re-quantified concentration with Qubit dsDNA HS assay  
** Colon tumor tissue, FFPE (CTT-FFPE)
** Colon tumor tissue, FFPE (CTT-FFPE)
* 2014-06-01, I added TE buffer to make conc. to 50ng/ul (total volume 40ul) based on original conc. and re-measured conc. with Qubit dsDNA HS assay again (1ul for the assay).
* 2014-06-01, I added TE buffer to make conc. to 50ng/ul (total volume 40ul) based on original conc. and re-measured conc. with Qubit dsDNA HS assay again (1ul for the assay).
* Dr. Zhang suggested to do two replicates for each level
* I will spike in 0.5% of unmethylated lambda DNA to check bisulfite conversion rate.
:{| {{table}} class = wikitable
:{| {{table}} class = wikitable
| align="center" style="width:120px;background:#f0f0f0;"|'''Sample'''
| align="center" style="width:120px;background:#f0f0f0;"|'''Sample'''
Line 18: Line 20:
| CTT-FFPE||76.00||26.32||40.00||13.68||64.8
| CTT-FFPE||76.00||26.32||40.00||13.68||64.8
|}
|}
==== Library preparation ====
==== Library preparation ====
* Low-input RRBS (5ng)
* Low-input RRBS (5ng)
Line 34: Line 35:
| CTT-FFPE||64.80||1.54||48.46
| CTT-FFPE||64.80||1.54||48.46
|}
|}
* For 5ng input, I will add 25pg of unmethylated lambda DNA (add 2.08ul of stock 12pg/ul)
* For 100ng input, I will add 0.5ng of unmethylated lambda DNA (add 0.5ul of stock 1ng/ul)
=== 1) DNA fragmentation with ''Msp''I ===
=== 1) DNA fragmentation with ''Msp''I ===
====1.1) Low-input RRBS ''Msp''I digestion set up ====
====1.1) Low-input RRBS ''Msp''I digestion set up ====
:{| {{table}} class = wikitable
| align="center" style="width:100px;background:#f0f0f0;"|'''CCT-5ng_1'''
| align="center" style="width:100px;background:#f0f0f0;"|'''CTT-5ng_2'''
| align="center" style="width:100px;background:#f0f0f0;"|'''CTT-F-5ng_1'''
| align="center" style="width:100px;background:#f0f0f0;"|'''CTT-F-5ng_2'''
| align="center" style="width:100px;background:#f0f0f0;"|'''5ng NTC'''
|-
|}
:{| {{table}} class = wikitable
:{| {{table}} class = wikitable
| align="center" style="width:120px;background:#f0f0f0;"|'''Sample'''
| align="center" style="width:120px;background:#f0f0f0;"|'''Sample'''
Line 42: Line 54:
| align="center" style="width:80px;background:#f0f0f0;"|'''10X Tango Buffer'''
| align="center" style="width:80px;background:#f0f0f0;"|'''10X Tango Buffer'''
| align="center" style="width:80px;background:#f0f0f0;"|'''MspI (10U/ul)'''
| align="center" style="width:80px;background:#f0f0f0;"|'''MspI (10U/ul)'''
| align="center" style="width:80px;background:#f0f0f0;"|'''12pg/ul unmeth-lambda DNA'''
| align="center" style="width:80px;background:#f0f0f0;"|'''H2O (ul)'''
| align="center" style="width:80px;background:#f0f0f0;"|'''H2O (ul)'''
| align="center" style="width:80px;background:#f0f0f0;"|'''Total'''
| align="center" style="width:80px;background:#f0f0f0;"|'''Total (ul)'''
|-
|-
| 1.1 CTT-frozen||2.00||2.5||2.00||1.00||12.50||18.00
| 1.1 CTT-frozen||2.00||2.5||2.00||1.00||2.08||10.42||18.00
|-
|-
| 1.2 CTT-FFPE||2.00||2.5||2.00||1.00||12.50||18.00
| 1.2 CTT-FFPE||2.00||2.5||2.00||1.00||2.08||10.42||18.00
|-
|-
| 1.3 NTC||0.00||2.5||2.00||1.00||12.50||18.00
| 1.3 NTC||0.00||2.5||2.00||1.00||2.08||10.42||18.00
|}
|}
:<u>'''''Msp''I reaction mix I'''</u>
:<u>'''''Msp''I reaction mix I'''</u>
:{| {{table}} border = 1
:{| {{table}} border = 1
| align="center" style="width:120px;background:#f0f0f0;"|'''Components'''
| align="center" style="width:150px;background:#f0f0f0;"|'''Components'''
| align="center" style="width:90px;background:#f0f0f0;"|'''1 rxn'''
| align="center" style="width:90px;background:#f0f0f0;"|'''1 rxn'''
| align="center" style="width:90px;background:#f0f0f0;"|'''3.5 rxn mix'''
| align="center" style="width:90px;background:#f0f0f0;"|'''5.5 rxn mix'''
|-
|-
| MspI (10U/ul)||1.00||3.50
| MspI (10U/ul)||1.00||5.50
|-
|-
| 10X Tango Buffer||2.00||7.00
| 10X Tango Buffer||2.00||11.00
|-
|-
| H2O||12.50||43.75
| 12pg/ul unmeth-lambda DNA||2.08||11.44
|-
| H2O||10.42||57.31
|-
|-
| Total||15.50||
| Total||15.50||
|}
|}
:- Aliquot 15.5ul to each tube
:- Aliquot 15.50ul to each tube
:- Add 2.5ul of diluted CTT DNA to each tube
:- Add 2.5ul of diluted CTT DNA to each tube
:- Mix by gentle pulse-vortexting for 10x (put reaction tube on PCR rack) and spin down
:- Mix by gentle pulse-vortexting for 10x (put reaction tube on PCR rack) and spin down
====1.2) STD RRBS ''Msp''I digestion set up ====
====1.2) STD RRBS ''Msp''I digestion set up ====
:{| {{table}} class = wikitable
| align="center" style="width:100px;background:#f0f0f0;"|'''CCT-100ng_1'''
| align="center" style="width:100px;background:#f0f0f0;"|'''CTT-100ng_2'''
| align="center" style="width:100px;background:#f0f0f0;"|'''CTT-F-100ng_1'''
| align="center" style="width:100px;background:#f0f0f0;"|'''CTT-F-100ng_2'''
| align="center" style="width:100px;background:#f0f0f0;"|'''100ng NTC'''
|-
|}
:{| {{table}} class = wikitable
:{| {{table}} class = wikitable
| align="center" style="width:120px;background:#f0f0f0;"|'''Sample'''
| align="center" style="width:120px;background:#f0f0f0;"|'''Sample'''
| align="center" style="width:80px;background:#f0f0f0;"|'''Conc. (ng/ul)'''
| align="center" style="width:80px;background:#f0f0f0;"|'''Conc. (ng/ul)'''
| align="center" style="width:80px;background:#f0f0f0;"|'''Volume for 100ng (ul)'''
| align="center" style="width:80px;background:#f0f0f0;"|'''Volume for 100ng (ul)'''
| align="center" style="width:80px;background:#f0f0f0;"|'''10X Tango Buffer'''
| align="center" style="width:80px;background:#f0f0f0;"|'''10X Tango Fuffer'''
| align="center" style="width:80px;background:#f0f0f0;"|'''''Msp''I (10U/ul)'''
| align="center" style="width:80px;background:#f0f0f0;"|'''MspI (10U/ul)'''
| align="center" style="width:80px;background:#f0f0f0;"|'''H2O to adjust volume (ul)'''
| align="center" style="width:80px;background:#f0f0f0;"|'''H2O to adjust volume (ul)'''
| align="center" style="width:80px;background:#f0f0f0;"|'''1ng/ul unmeth-lambda DNA'''
| align="center" style="width:80px;background:#f0f0f0;"|'''H2O in MspI mix(ul)'''
| align="center" style="width:80px;background:#f0f0f0;"|'''H2O in MspI mix(ul)'''
| align="center" style="width:80px;background:#f0f0f0;"|'''Total (ul)'''
|-
|-
| 1.1 CTT-frozen||52.30||1.91||2.00||1.00||5.09||10.00
| 1.1 CTT-frozen||52.30||1.91||2.00||1.00||5.09||0.50||7.50||18.00
|-
|-
| 1.2 CTT-FFPE||64.80||1.54||2.00||1.00||5.46||10.00
| 1.2 CTT-FFPE||64.80||1.54||2.00||1.00||5.46||0.50||7.50||18.00
|-
|-
| 1.3 NTC||0.00||0.00||2.00||1.00||7.00||10.00
| 1.3 NTC||0.00||0.00||2.00||1.00||7.00||0.50||7.50||18.00
|}
|}
:<u>'''''Msp''I reaction mix II'''</u>
:<u>'''''Msp''I reaction mix II'''</u>
:{| {{table}} border = 1
:{| {{table}} border = 1
| align="center" style="width:120px;background:#f0f0f0;"|'''Components'''
| align="center" style="width:150px;background:#f0f0f0;"|'''Components'''
| align="center" style="width:90px;background:#f0f0f0;"|'''1 rxn'''
| align="center" style="width:90px;background:#f0f0f0;"|'''1 rxn'''
| align="center" style="width:90px;background:#f0f0f0;"|'''3.5 rxn mix'''
| align="center" style="width:90px;background:#f0f0f0;"|'''5.5 rxn mix'''
|-
| MspI (10U/ul)||1.00||5.50
|-
|-
| ''Msp''I (10U/ul)||1.00||3.50
| 10X Tango Buffer||2.00||11.00
|-
|-
| 10X Tango Buffer||2.00||7.00
| 1ng/ul unmeth-lambda DNA||0.5||2.75
|-
|-
| H2O||10.00||35.00
| H2O||10.00||55.00
|-
|-
| Total||13.00||
| Total||13.50||
|}
|}
:- Add H2O to adjust volume as table above
:- Add H2O to adjust volume as table above
:- Aliquot 13ul to each tube
:- Aliquot 13.5ul to each tube
:- Add  CTT DNA to each tube following the number in table above
:- Add  CTT DNA to each tube following the number in table above
:- Mix by gentle pulse-vortexting for 10x (put reaction tube on PCR rack) and spin down
:- Mix by gentle pulse-vortexting for 10x (put reaction tube on PCR rack) and spin down

Revision as of 17:52, 2 June 2014

RRBS library preparation of colon tumor tissue samples from Biochain

Sample list and details

  • Sample type: genomic DNA from primary tumor tissue
    • Colon tumor tissue, frozen (CTT-frozen): re-quantified concentration with Qubit dsDNA HS assay
    • Colon tumor tissue, FFPE (CTT-FFPE)
  • 2014-06-01, I added TE buffer to make conc. to 50ng/ul (total volume 40ul) based on original conc. and re-measured conc. with Qubit dsDNA HS assay again (1ul for the assay).
  • Dr. Zhang suggested to do two replicates for each level
  • I will spike in 0.5% of unmethylated lambda DNA to check bisulfite conversion rate.
Sample Original conc. (ng/ul) Estimate volume (ul) Dilute to 50ng/ul (total volume) Add TE buffer (ul) Qubit conc. (ng/ul)
CTT-Frozen 580.00 3.45 40.00 36.55 52.3
CTT-FFPE 76.00 26.32 40.00 13.68 64.8

Library preparation

  • Low-input RRBS (5ng)
  • Standard RRBS (100ng)

Experimental procedures

  • I firstly dilute CTT gDNA to 2ng/ul volume 50ul
Sample Conc. (ng/ul) Stock volume (ul) TE (ul)
CTT-frozen 52.30 1.91 48.09
CTT-FFPE 64.80 1.54 48.46
  • For 5ng input, I will add 25pg of unmethylated lambda DNA (add 2.08ul of stock 12pg/ul)
  • For 100ng input, I will add 0.5ng of unmethylated lambda DNA (add 0.5ul of stock 1ng/ul)

1) DNA fragmentation with MspI

1.1) Low-input RRBS MspI digestion set up

CCT-5ng_1 CTT-5ng_2 CTT-F-5ng_1 CTT-F-5ng_2 5ng NTC
Sample Conc. (ng/ul) Volume for 5ng (ul) 10X Tango Buffer MspI (10U/ul) 12pg/ul unmeth-lambda DNA H2O (ul) Total (ul)
1.1 CTT-frozen 2.00 2.5 2.00 1.00 2.08 10.42 18.00
1.2 CTT-FFPE 2.00 2.5 2.00 1.00 2.08 10.42 18.00
1.3 NTC 0.00 2.5 2.00 1.00 2.08 10.42 18.00
MspI reaction mix I
Components 1 rxn 5.5 rxn mix
MspI (10U/ul) 1.00 5.50
10X Tango Buffer 2.00 11.00
12pg/ul unmeth-lambda DNA 2.08 11.44
H2O 10.42 57.31
Total 15.50
- Aliquot 15.50ul to each tube
- Add 2.5ul of diluted CTT DNA to each tube
- Mix by gentle pulse-vortexting for 10x (put reaction tube on PCR rack) and spin down

1.2) STD RRBS MspI digestion set up

CCT-100ng_1 CTT-100ng_2 CTT-F-100ng_1 CTT-F-100ng_2 100ng NTC
Sample Conc. (ng/ul) Volume for 100ng (ul) 10X Tango Fuffer MspI (10U/ul) H2O to adjust volume (ul) 1ng/ul unmeth-lambda DNA H2O in MspI mix(ul) Total (ul)
1.1 CTT-frozen 52.30 1.91 2.00 1.00 5.09 0.50 7.50 18.00
1.2 CTT-FFPE 64.80 1.54 2.00 1.00 5.46 0.50 7.50 18.00
1.3 NTC 0.00 0.00 2.00 1.00 7.00 0.50 7.50 18.00
MspI reaction mix II
Components 1 rxn 5.5 rxn mix
MspI (10U/ul) 1.00 5.50
10X Tango Buffer 2.00 11.00
1ng/ul unmeth-lambda DNA 0.5 2.75
H2O 10.00 55.00
Total 13.50
- Add H2O to adjust volume as table above
- Aliquot 13.5ul to each tube
- Add CTT DNA to each tube following the number in table above
- Mix by gentle pulse-vortexting for 10x (put reaction tube on PCR rack) and spin down

- Incubate at 37C for 3hr
- Heat inactivate at 65C for 20min