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|| | | 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|| | | 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|| | | 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: | | 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;"|''' | | align="center" style="width:90px;background:#f0f0f0;"|'''5.5 rxn mix''' | ||
|- | |- | ||
| MspI (10U/ul)||1.00|| | | MspI (10U/ul)||1.00||5.50 | ||
|- | |- | ||
| 10X Tango Buffer||2.00|| | | 10X Tango Buffer||2.00||11.00 | ||
|- | |- | ||
| H2O|| | | 12pg/ul unmeth-lambda DNA||2.08||11.44 | ||
|- | |||
| H2O||10.42||57.31 | |||
|- | |- | ||
| Total||15.50|| | | Total||15.50|| | ||
|} | |} | ||
:- Aliquot 15. | :- 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 | | align="center" style="width:80px;background:#f0f0f0;"|'''10X Tango Fuffer''' | ||
| align="center" style="width:80px;background:#f0f0f0;"|''' | | 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|| | | 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|| | | 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|| | | 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: | | 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;"|''' | | align="center" style="width:90px;background:#f0f0f0;"|'''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|| | | H2O||10.00||55.00 | ||
|- | |- | ||
| Total||13. | | Total||13.50|| | ||
|} | |} | ||
:- Add H2O to adjust volume as table above | :- Add H2O to adjust volume as table above | ||
:- Aliquot | :- 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