Noi/NOTES/2014-6-2: Difference between revisions
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:- Incubate at 37C for 3hr | :- Incubate at 37C for 3hr | ||
:- Heat inactivate at 65C for 20min | :- 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) <u>no heat lid will help to pretech Klenow fragment, exo</u> | |||
:- 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'''<br> | |||
{| {{table}}class = wikitable | |||
| align="center" style="width:80px;background:#f0f0f0;"|'''CCT-5ng_1''' | |||
| align="center" style="width:80px;background:#f0f0f0;"|'''CTT-5ng_2''' | |||
| align="center" style="width:80px;background:#f0f0f0;"|'''CTT-F-5ng_1''' | |||
| align="center" style="width:80px;background:#f0f0f0;"|'''CTT-F-5ng_2''' | |||
| align="center" style="width:80px;background:#f0f0f0;"|'''5ng NTC''' | |||
|- | |||
| Ind_9||Ind_10||Ind_11||Ind_13||Ind_14 | |||
|} | |||
{| {{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''' | |||
|- | |||
| Ind_15||Ind_16||Ind_18||Ind_19||Ind_20 | |||
|} | |||
'''''<span style="color:crimson"><u>Prep</u></span>'''''<br> | |||
- Diluted 1:10 of TrueSeq methylated adaptors by mixing 1ul of adaptor with 10ul H2O and mix<br> | |||
- Prepare ligation reaction mix | |||
:{| {{table}} class = wikitable | |||
| align="center" style="background:#f0f0f0;"|'''Components''' | |||
| align="center" style="background:#f0f0f0;"|'''Volume (ul)''' | |||
| align="center" style="background:#f0f0f0;"|'''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'' |
Revision as of 22:12, 2 June 2014
RRBS library preparation of colon tumor tissue samples from Biochain
- [Link to calendar]
- References
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 pretech 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