Noi/NOTES/2014-10-31

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Repeating of BIS/DNA SMART ChIP-Seq protocol

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  • According to experiment on 2014-10-29, I still see amplification of contaminants in BIS-NTC. In that experiment, I used BisulFlash DNA Methylation kit (bead-base) from Epigentek. For this experiment, I would ignore a bisulfite conversion rate and recovery yield of this kit. I just wanted to try the new kit with the price is affordable to see whether I can remove contamination. Even the contamination was observed, the level was lower than Zymo DNA Methylation-Lightning kit (column-based).
  • In this experiment, I want to repeat experiment again using Zymo DNA Methylation-Lightning Magprep kit. I want to see if the level of contamination is comparable to the first experiment that I tried with DNA Methylation-Lightning column-based kit in the first experiment on 2014-10-08. This might explain about contamination in CT conversion reagent and the procedures between column and bead.
  • I also wanted to compare the yield after amplification between BIS-100ng input and BIS-NTC.
  • I used the old "Zymo DNA Methylation-Lightning Magprep kit" I purchased in September 2013 as getting the new kit is too expensive for trial experiment. However, I believe that the reagents should be clean. I UV all reagent except conversion reagent.
  • I used ClonTech control fragmented human gDNA for this experiment. I followed the procedures of bisulfite conversion by zymo DNA Methylation-Lightning Magprep kit and resuspend with 22ul Elution buffer. Then transfer 20ul of converted DNA for library preparation
  • I used the same procedure of library preparation using ClonTech DNA SMRT-Seq kit as describe in [2014-10-29]

DNA SMRT ChIP-Seq library preparation

A: Denaturation of DNA

Template Input amount BIS-CVT Stock conc. Volume (ul) Dilution buffer
Control fragmented DNA 1ng YES NA 20.00 0.00
Control fragmented DNA 100pg YES NA 20.00 0.00
NTC NA YES NA 20.00 0.00
NTC NA YES NA 20.00 0.00
Control fragmented DNA 100pg no 50pg/ul 2.00 18.00
NTC NA no NA 0.00 20.00
- 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 6.2 rxn Dephosphorylation Master Mix for all reactions by combining the following reagents on ice.
Components 1rxn (ul) 6.2 rxn
DNA SMART Buffer 3.25 20.15
Shrimp Alkaline Phosphatase 0.75 4.65
Total 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 7.2rxn T-Tailing Master Mix for all reactions by combining the following reagents on ice
Components 1rxn (ul) 6.2 rxn
DNA SMART T-Tailing Mix 1.00 6.2
Terminal Deoxynucleotidyl Transferase 1.00 6.2
Total 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 6.2rxn Template Switching Master Mix for all reactions by combining the following reagents on ice.
Components 1rxn (ul) 6.2 rxn
DNA SMART Buffer 6.00 37.20
DNA SMART Oligonucleotide mix 6.00 37.20
SMARTScribe Reverse Transcriptase 4.00 24.80
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

  • I would initially do a quick qPCR to test the PCR cycle number