Noi/NOTES/2014-6-29: Difference between revisions

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:- Spin down column at 12,000 rpm for 1min
:- Spin down column at 12,000 rpm for 1min
:- Continue to amplification (During elution step, PCR mix should be prepared)
:- Continue to amplification (During elution step, PCR mix should be prepared)
== Amplification ==
=== TEST PCR ===
'''Colon tumor samples'''
[[File:ZhangLab_2 2014-06-30 17hr 16min_6T_100ngRRBS_TEST-14X.jpg| 520px]]
 
'''Lung tumor samples'''
[[File:ZhangLab_2 2014-06-30 17hr 16min_7T_100ngRRBS_TEST-14X-e.jpgg| 520px]]
* I do not know why sample 7-T_3.2 and 7-T_4.1 failed. May need to check with Dr. Zhang if he want to sequence the two duplicates or only 1 library for each sample.

Revision as of 03:13, 1 July 2014

Preparation of RRBS sequencing libraries of plasma DNA from colon and lung cancer patients

Link to calendar

Sample info

Project ID Zhang lab ID Conc. (ng/ul)
UCSD-006.1 6-P_1 0.200
UCSD-006.2 6-P_2 1.100
UCSD-006.3 6-P_3 1.570
UCSD-006.4 6-P_4 0.889
UCSD-006.5 6-P_5 0.487
UCSD-006.6 6-P_6 0.249
UCSD-006.7 6-P_7 0.247
UCSD-006.8 6-P_8 0.226
UCSD-006.9 6-P_9 0.408
UCSD-006.10 6-P_10 0.333
Project ID Zhang lab ID Conc. (ng/ul)
UCSD-007.1 7-P_1 0.692
UCSD-007.2 7-P_2 1.140
UCSD-007.3 7-P_3 0.208
UCSD-007.4 7-P_4 0.202
UCSD-007.5 7-P_5 1.110
UCSD-007.6 7-P_6 0.233
UCSD-007.7 7-P_7 0.229
UCSD-007.8 7-P_8 0.167
UCSD-007.9 7-P_9 0.231
UCSD-007.10 7-P_10 0.203
  • X-Y_Z:
    • X = project ID number. 4 = pancreas, 6 = colon, 7 = lung
    • Y = sample type. T = tissue, P = plasma, S = serum
    • Z = sample ID

1) MspI digestion

  • Note that the protocol of low-input DNA (1-5ng) will be different from the STD RRBS protocol (100ng).
  • Conditions: Digest 1ng of DNA with 10U of MspI in 18ul reaction at 37C O/N (normally 3h) and heat inactivate at 65C 20min
  • For 1ng DNA input, I used 1ul (10U) of MspI.
  • I spike in 1% of unmeth lambda DNA (10pg)
  • I actually start MspI at late night of 2014-06-29, so I incubated it O/N as it doesn't hurt to incubate DNA with MspI longer than 3h.


Msp I Reaction Mix

Components 1x rxn 26 rxn Mix
MspI (10U/ul) 1.00 26.00
10x Tango Buffer 2.00 52.00
12pg/ul unmeth-lambda DNA 0.83 21.67
H2O 8.17 212.33
Total 12.00

Prep
- Aliquot 39ul of MspI reaction mix to 8-tube strip.

- Add H2O to each tube
- Add 1ng of DNA to each tube
- Add 12ul of MspI reaction Mix to each tube and pipette few times
- Spin down the tubes
- Mix by gentle pulse-vortexing on PCR rack 10x
- Spin down the tubes
- 37C, O/N (~12.5h) --> 65C, 20min --> hold at 4C (set heat lid)


2) End-repair/dA-tailing

Prep
- Mix 29ul of Klenow exo- with 29ul of dA:dC:dG (stock 20mM:2mM:2mM) just right before adding to MspI digested DNA
- Aliquot 7ul of Klenow, exo- and dA:dC:dG mix to 8-tube strip.

- Add 2ul of Klenow, exo- and dA:dC:dG mix to each tube of MspI digested DNA with multichannel pipette. Pipette few times
- Spin down the tube
- Mix by gentle pulse-vortexing on PCR rack 10x
- Spin down the tube
- 30C, 20min --> 37C, 20min --> hold at 4C (no heat lid)
- 75C, 10min --> hold at 4C (heat lid on)


3) Methylated adaptor ligation

  • Conditions: Add 1ul of 1:20 diluted TruSeq adaptors (do not know actual concentration of adaptors) in 25ul ligation reaction with 6000U of T4 DNA ligase

Ligation reaction set up

Prep
- Thaw diluted TruSeq adaptor on ice (used the diluted adaptors prepared on 2014-06-20, keep it on ice all the time)
- Prepare ligation reaction mix

Ligation reaction Mix

Components 1x rxn 26x rxn
dA-tailed DNA 20.00 0.00
10X Tango buffer 0.50 13.00
HC T4 DNA ligase (30units/ul) 1.00 26.00
10mM ATP 1.25 32.50
H2O 1.25 32.50
Total 24.00

Prep
- Aliquot 13ul of ligation reaction mix to 8-tube strip.

- Add 1ul of 1:20 diluted TruSeq adaptors and pipette few times to make sure adaptors were added to the reaction
Sample TruSeq Index Sample TruSeq Index
6-P_1 13 7-P_1 1
6-P_2 14 7-P_2 2
6-P_3 15 7-P_3 4
6-P_4 16 7-P_4 5
6-P_5 27 7-P_5 6
6-P_6 18 7-P_6 7
6-P_7 19 7-P_7 8
6-P_8 20 7-P_8 10
6-P_9 21 7-P_9 11
6-P_10 22 7-P_10 12
6-P_NTC1 22 7-P_NTC1 12
6-P_NTC2 22 7-P_NTC2 12
- Add 4ul of ligation reaction mix with multichannel pipette
- Spin down the tube
- Mix by gentle pulse-vortexing on PCR rack 10x
- Spin down the tube
- 16C, 20h (no heat lid. Ligation time is flexible. I usually do at least 20h at 16C)
- Mix by gentle pulse-vortexing on PCR rack 5x
- Quickly spin down
- Heat inactivate at 65C, 20min --> hold at 4C (heat lid on)

Bisulfite conversion

  • Performed bisulfite conversion using LifeTech MethylCode™ Bisulfite Conversion Kit and did the same procedure following manufacturer's instruction and elute with 38ul Elution buffer.

Prep

- Prepare 2 tubes of complete CT Conversion Reagent by adding 800ul H2O, 50ul Resuspension Buffer, and 300ul Dilution Buffer
- Mix well and spin down the tube
- Put on thermomixer at 37C for 15min, 1,000rpm
- Solution should be clear
- Spin down the tube
- Add 120ul complete CT Converson Reagent to 30ul bead-purified adaptor ligated DNA --> total volume 150ul
- Mix by pipetting 10X with multi-channel pipette
- Spin down the tube
- Incubate following below program
- 98°C for 10 minutes (DNA denaturation)
- 64°C for 2.5 hours (Bisulfite conversion)
- 4°C storage for up to 20 hours or continue to desulfonation
- Add 600ul Binding Buffer to each column
- Adjust p200 to 200ul and transfer bisulfite treated DNA to the column
- Mix by pipetting 5x
- Spin down column at 14,000 rpm for 30s
- Discard spnt
- Add 100ul Wash Buffer (Aliquot Wash Buffer in 5mL tube)
- Spin down column at 14,000 rpm for 30s (do not discard spnt)
- Add 200ul Desulfonation Buffer (aliquot Desulfonation Buffer in 2mL or 5mL tube)
- Spin down column at 14,000 rpm for 30s (do not discard spnt)
- Add 200ul Wash Buffer
- Spin down column at 14,000 rpm for 30s
- Discard spnt
- Add 200ul Wash Buffer
- Spin down column at 14,000 rpm for 3min (do not discard spnt)
- Transfer column to prepared 1.5mL LoBind tube & make sure that column does not touch spnt in the collecting tube before transferring to new tube
- Add 38ul of Elution Buffer (warm Elution Buffer at 60C for 10min)
- Wait for 2min
- Spin down column at 12,000 rpm for 1min
- Continue to amplification (During elution step, PCR mix should be prepared)