Noi/NOTES/2014-6-2

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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 41.25
Total 11.00
- Add H2O to adjust volume as table above
- Aliquot 11ul 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

3) End-repair/dA-tailing

  • To prevent contamination to enzyme tube by multiple time pipetting, I aliquot 11ul each of Klenow fragment, exo- and dA:dC:dG mix into PCR tube.
- Add 2ul of dA:dC:dG/Klenow fragment exo- to each tube
- Spin down the tube
- Mix by gentle pulse-vortexting for 10x
- Spin down the tube
- Incubate at 30C for 20min (for gap-filling) --> 37C for 20min (for extra dA-tailing) no heat lid will help to protect Klenow fragment, exo
- Heat inactivate enzyme at 75C for 10min
- Set program to hold at 4C
- Spin down the tube before continuing to next step

Methylated adaptor ligation

  • To avoid the overlapping of indexes to some scRRBS libraries that may need to sequence in the same run (HiSeq Rapid run has only 2 lanes), I will not use index 2, 4, 5, and 12
  • I will use 1ul of 1:10 diluted TruSeq adaptors for both low-input RRBS and STD RRBS.

Index list

CCT-5ng_1 CTT-5ng_2 CTT-F-5ng_1 CTT-F-5ng_2 5ng NTC
Ind_9 Ind_10 Ind_11 Ind_13 Ind_14
CCT-100ng_1 CTT-100ng_2 CTT-F-100ng_1 CTT-F-100ng_2 100ng NTC
Ind_15 Ind_16 Ind_18 Ind_19 Ind_20

Prep
- Diluted 1:10 of TrueSeq methylated adaptors by mixing 1ul of adaptor with 10ul H2O and mix
- Prepare ligation reaction mix

Components Volume (ul) 11x rxn mix
dA-tailed reaction 20.00 0.00
10X Tango buffer 0.50 5.50
HC T4 DNA ligase (30units/ul) 1.00 11.00
10mM ATP 1.25 13.75
H2O 1.25 13.75
Total 24.00 44.00
- Add 1ul of diluted methylated adapter
- Add 4ul of ligation reaction mix
- Spin down the tube
- Mix by gentle pulse-vortexting for 10x
- Spin down the tube
- Incubate at 16C for 16h (no heat lid)
- Heat inactivate at 65C for 20min