Noi/NOTES/2014-6-2
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RRBS library preparation of colon tumor tissue samples from Biochain
Sample list
- Sample type: genomic DNA
- 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).
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
1) DNA fragmentation with MspI
1.1) Low-input RRBS MspI digestion set up
Sample Conc. (ng/ul) Volume for 5ng (ul) 10X Tango Buffer MspI (10U/ul) H2O (ul) Total 1.1 CTT-frozen 2.00 2.5 2.00 1.00 12.50 18.00 1.2 CTT-FFPE 2.00 2.5 2.00 1.00 12.50 18.00 1.3 NTC 0.00 2.5 2.00 1.00 12.50 18.00
- MspI reaction mix I
Components 1 rxn 3.5 rxn mix MspI (10U/ul) 1.00 3.50 10X Tango Buffer 2.00 7.00 H2O 12.50 43.75 Total 15.50
- - Aliquot 15.5ul 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
Sample Conc. (ng/ul) Volume for 100ng (ul) 10X Tango Buffer MspI (10U/ul) H2O to adjust volume (ul) H2O in MspI mix(ul) 1.1 CTT-frozen 52.30 1.91 2.00 1.00 5.09 10.00 1.2 CTT-FFPE 64.80 1.54 2.00 1.00 5.46 10.00 1.3 NTC 0.00 0.00 2.00 1.00 7.00 10.00
- MspI reaction mix II
Components 1 rxn 3.5 rxn mix MspI (10U/ul) 1.00 3.50 10X Tango Buffer 2.00 7.00 H2O 10.00 35.00 Total 13.00
- - Add H2O to adjust volume as table above
- - Aliquot 13ul 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