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