Noi/NOTES/2014-10-8: Difference between revisions

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'''NOTE:''' Add the Shrimp Alkaline Phosphatase to the buffer immediately prior to use. Mix well by
gently vortexing and spin the tubes briefly in a microcentrifuge. Keep the DNA SMART Buffer on
ice until its next use in Step V.D.
:- Add 4 μl of the Dephosphorylation Master Mix to each reaction tube from Step V.A. Mix the contents of the tubes by gently vortexing, and spin the tubes briefly"
:- Place the tubes in a preheated thermal cycler and run program ChIP-B. Leave the tubes at 4°C until the next step<br>
:- '''ChIP-C''':      37°C 20 min --> 70°C 10 min -- > 4°C hold<br>

Revision as of 21:58, 10 October 2014

Integration of DNA SMART ChIP-Seq protocol with RRBS for low input DNA methylation assay

Link to calendar

  • Dr. Zhang suggested to try template switching protocol in stead of the standard adaptor ligation base method to generate bisulfite sequencing library. The idea is to fragment DNA by MspI to enrich only the targets for RRBS then perform bisulfite conversion on MspI-digested DNA. The single strand bisulfite converted DNA will be used as the template for the subsequent steps, including dephosphorylation, poly dT-tailng, first strand cDNA synthesis, and template switching to append the partial adaptor sequence to the newly synthesized cDNA. Then PCR with adaptor primers will be performed to amplified the libraries. One advantage of performing bisulfite conversion before generating sequencing library is that the template with ligated adaptors won't be damaged within the molecules as occurs in standard WGBS protocol.

DNA SMART ChIP-Seq kit Manual

TEST #1

  • I used gDNA sample of cancer cell line CANC1 (eq. to XXXX) for the first TEST experiment before moving to gDNA from solid tumor tissue.
  • Vary amount of template at 1ng, 100pg and 10pg.
- Dilute 86.4ng/ul to 2ng/ul --> re-quantify conc. by Qubit: 1.57ng/ul
- Mix 2.32ul of 86.4ng/ul of CANC1 and 97.68ul of TE buffer
- Dilute to : 100pg/ul, 10pg/ul, 1pg/ul to used 10ul of each concentration for MspI digestion

MspI reaction Mix

Components 1x rxn 4.5x rxn
MspI (10U/ul) 1.00 4.50
10x Tango Buffer 2.00 9.00
H2O 7.00 31.50
Total 10.00

MspI digestion set up

Input amount Stock used (pg/ul) Volume of stock (ul) MspI reaction mix Total (ul)
1ng 100.00 10.00 10.00 20.00
100pg 10.00 10.00 10.00 20.00
10pg 1.00 10.00 10.00 20.00
NTC H2O 10.00 10.00 20.00
- Add 10ul of DNA to each tube
- Add 10ul 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, 3h --> 65C, 20min --> hold at 4C (set heat lid)


- Perform bisulfite conversion by EZ-Lightning DNA Methylation kit without using tRNA as carrier.
- Elute with 12ul of Elution Buffer. The left over volume of DNA is ~10ul.

Note from DNA SMART ChIP-Seq kit

Sample Requirements
This kit can generate sequencing libraries from 100 pg–10 ng of ssDNA or dsDNA up to 2 kb in length, but it has been optimized specifically for ChIP DNA with an average size of 200–400 bp. If your starting material is significantly larger, you may need to increase the number of PCR cycles to obtain sufficient yield after size selection Starting material should be in 1–20 µl of an alkaline, low ionic buffer such as the DNA Dilution Buffer (5 mM Tris-HCl pH 8.5) provided, or water. The optimal pH range of samples is 7.5–8.5. Avoid EDTA-containing buffers


A: Denaturation of DNA

- Aliquot appropriate amounts of DNA (100 pg to 10 ng) in a maximum volume of 20 µl in PCR tubes.
I add 10ul of Dilution buffer to 10ul of eluted bisulfite converted DNA and transfer to 0.2mL reaction tube and spin down.
- Incubate the tubes at 94°C in a preheated, hot-lid thermal cycler (ChIP-94) for exactly 2 min
- Immediately remove the samples and place on ice for at least 2 minutes
- Spin the tubes briefly to bring down any condensation.

B: Dephosphorylation of 3’ Ends

- Prepare enough Dephosphorylation Master Mix for all reactions, plus 10%, by combining the following reagents on cool box.
Components 1x rxn 4.4 rxn
DNA SMART Buffer (red cap) 3.25 14.30
Shrimp Alkaline Phosphatase (red cap) 0.75 3.30
Total volume per reaction 4.00
NOTE: Add the Shrimp Alkaline Phosphatase to the buffer immediately prior to use. Mix well by 
gently vortexing and spin the tubes briefly in a microcentrifuge. Keep the DNA SMART Buffer on 
ice until its next use in Step V.D.
- Add 4 μl of the Dephosphorylation Master Mix to each reaction tube from Step V.A. Mix the contents of the tubes by gently vortexing, and spin the tubes briefly"
- Place the tubes in a preheated thermal cycler and run program ChIP-B. Leave the tubes at 4°C until the next step
- ChIP-C: 37°C 20 min --> 70°C 10 min -- > 4°C hold