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  [[File:ZhangLab_2 2014-10-09 13hr 40min_10pg-20-24x_NTC23-24x.jpg| 400px]]
  [[File:ZhangLab_2 2014-10-09 13hr 40min_10pg-20-24x_NTC23-24x.jpg| 400px]]
=== Discussion (will be added) ===
<br>
<br>
== <u>TEST #2</u> ==
'''2014-10-09'''<br>
* From previous experiment, I got amplified in NTC at the same position of the reaction with template DNA. Even the amplified level in NTC was lower than 1ng & 100pg input DNA, this seem to be very high. I did not see the different between 10pg input and NTC.
* This might be contamination. I need to figure out where this signal from.
* Actually, I should have included '''positive control''' using the fragmented DNA that came with the kit when I set up the reaction in step A: Dephosphorylation and I should also have set up another NTC in step A. This will help to identify that where did the contamination came from. It could be the step of MspI digestion and bisulfite conversion or since step A of the protocol.

Revision as of 00:32, 11 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)


Bisulfite conversion

- Perform bisulfite conversion by EZ-Lightning DNA Methylation kit using instructions provided by manufacturer without using tRNA as carrier.
- Elute with 12ul of Elution Buffer. The left over volume of DNA is ~10ul.
- Continue to the the steps of DNA SMART ChIP-Seq protocol

<ve> 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-B: 37°C 10 min --> 65°C 5 min --> 4°C hold
NOTE: During the 65°C incubation step, prepare the T-Tailing Master Mix in Step V.C.1 below.


C: T-Tailing

- Prepare enough T-Tailing Master Mix for all reactions, plus 10%, by combining the following reagents on ice
Components 1x rxn 4.2 rxn
DNA SMART T-Tailing Mix (green cap) 1.00 4.20
Terminal Deoxynucleotidyl Transferase (green cap) 1.00 4.20
Total volume per reaction 2.00
- Add 2 μl of the T-Tailing Master Mix to each reaction tube. 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-C. Leave the tubes at 4°C until the next step.
- ChIP-C: 37°C 20 min --> 70°C 10 min -- > 4°C hold

D: Primer Annealing and Template Switching Reaction

The DNA SMART Poly(dA) Primer anneals to the ssDNA template followed by DNA replication and 
template switching. At the end of this step, the DNA is double stranded with partial adapters on both 
ends.

NOTE: Prepare the Template Switching Master Mix (Step V.D.5) prior to starting the primer annealing 
step below.
- Primer Annealing: Add 2 μl of the DNA SMART Poly(dA) Primer (yellow cap) to each reaction tube. Mix the contents of the tubes by gently vortexing and spin the tubes briefly."
- Incubate the tubes at 94°C in a preheated, hot-lid thermal cycler (ChIP-94) for exactly 1 min.
- Immediately remove the samples and place on ice for at least 2 minutes.
- Spin the tubes briefly to bring down any condensation.
NOTE: Proceed immediately to the next step
- Prepare enough Template Switching Master Mix for all reactions, plus 10%, by combining the following reagents on ice.
Components 1rxn (ul) 4.4 rxn
DNA SMART Buffer 6.00 26.40
DNA SMART Oligonucleotide mix 6.00 26.40
SMARTScribe Reverse Transcriptase 4.00 17.60
Total 16.00
Note: DO NOT add the SMARTScribe Reverse Transcriptase to the buffer until immediately prior to use.
Mix well by gently vortexing and spin the tubes briefly in a micro centrifuge
- DNA Replication and Template Switching: Add the SMARTScribe Reverse Transcriptase (purple cap) to the Template Switching Master Mix prepared in Step V.D.5, then add 16 μl of the Template Switching Master Mix to each reaction tube. 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-D. Leave the tubes at 4°C until the next step
- ChIP-D: 42°C 90 min --> 70°C 15 min -- > 4°C hold
*Note: I left the reaction O/N at 4C before continuing to PCR.

E. ChIP-Seq Library Amplification by PCR

Result

File:ZhangLab 2 2014-10-09 13hr 31min 1ng-15-20x-NTC15-18x.jpg

File:ZhangLab 2 2014-10-09 14hr 11min 100pg-18-22x NTC19-22.jpg

File:ZhangLab 2 2014-10-09 13hr 40min 10pg-20-24x NTC23-24x.jpg

Discussion (will be added)



TEST #2

2014-10-09

  • From previous experiment, I got amplified in NTC at the same position of the reaction with template DNA. Even the amplified level in NTC was lower than 1ng & 100pg input DNA, this seem to be very high. I did not see the different between 10pg input and NTC.
  • This might be contamination. I need to figure out where this signal from.
  • Actually, I should have included positive control using the fragmented DNA that came with the kit when I set up the reaction in step A: Dephosphorylation and I should also have set up another NTC in step A. This will help to identify that where did the contamination came from. It could be the step of MspI digestion and bisulfite conversion or since step A of the protocol.