Noi/NOTES/2014-10-29: Difference between revisions

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=== B: Dephosphorylation of 3’ Ends ===
=== B: Dephosphorylation of 3’ Ends ===
:- Prepare 7.2 rxn Dephosphorylation Master Mix for all reactions.
:- Prepare 7.2 rxn Dephosphorylation Master Mix for all reactions by combining the following reagents on ice.
::{| {{table}}
::{| {{table}}
| align="center" style="width:240px;background:#f0f0f0;"|'''Components'''
| align="center" style="width:240px;background:#f0f0f0;"|'''Components'''
Line 82: Line 82:
:- '''ChIP-B:'''      37°C 10 min --> 65°C 5 min --> 4°C hold<br>
:- '''ChIP-B:'''      37°C 10 min --> 65°C 5 min --> 4°C hold<br>
  '''NOTE:''' During the 65°C incubation step, prepare the T-Tailing Master Mix in Step V.C.1 below.
  '''NOTE:''' During the 65°C incubation step, prepare the T-Tailing Master Mix in Step V.C.1 below.
<br>
=== C: T-Tailing ===
:- Prepare 7.2rxn T-Tailing Master Mix for all reactions by combining the following reagents on ice
::{| {{table}}
| align="center" style="width:240px;background:#f0f0f0;"|'''Components'''
| align="center" style="width:100px;background:#f0f0f0;"|'''1rxn (ul)'''
| align="center" style="width:100px;background:#f0f0f0;"|'''7.2 rxn'''
|-
| DNA SMART T-Tailing Mix||1.00||7.2
|-
| Terminal Deoxynucleotidyl Transferase||1.00||7.2
|-
| 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<br>
<br>
<br>

Revision as of 19:54, 29 October 2014

Generation of sequencing library of a customized SeqCap EZ Epi Probe Pool

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  • 2014-10-29: I am expecting the package of a customized SeqCap EZ Epi probe pool that Dr. Zhang ordered for target enrichment of the blood UMRs from all WGBS libraries
  • Probe information:
    • Design name: 140923_HG19_UMR_v1_Epi_HX1
    • Volume: 54ul


Things to do upon receive the probe pool

1. Recommended by instruction in CHAPTER2:

Upon receipt, undertake the following steps to ensure the highest performance of the SeqCap Epi probe pool to avoid multiple freeze/thaw cycles or potential accidental contamination:
  1. If frozen, thaw the tube of SeqCap Epi probe pool on ice.
  2. Vortex the SeqCap Epi probe pool for 3 seconds.
  3. Centrifuge the tube of SeqCap Epi probe pool at 10,000 x g for 30 seconds to ensure that the liquid is at the bottom of the tube before opening the tube.
  4. Aliquot the SeqCap Epi probe pool into single-use aliquots (4.5 µl/aliquot) in 0.2 ml PCR tubes (or 96-well plates if following the higher throughput protocol described in Appendix C) and store at -15 to -25°C until use. The presence of some residual volume after dispensing all single-use aliquots is normal.
  5. When ready to perform the experiment, thaw the required number of single-use SeqCap Epi probe pool aliquots on ice.

NOTE: The SeqCap Epi probe pool should not undergo multiple freeze/thaw cycles. To help ensure the highest performance of the SeqCap Epi probe pool, Roche NimbleGen recommends aliquoting the SeqCap Epi probe pool into single-use volumes to prevent damage from successive freeze/thaw cycles.

Label PCR tube: Hg19 UMR v1 141029
Location: -20C, in original box within Noi Library prep kit box. 
I got total 16 tubes of 4.5ul aliquot. The instruction said it's normal to get extra volume of the probe.

2. For sequencing library preparation

  1. Take a small aliquot the left over of the probe pool after a single-use aliquots for 12 rxn for library prep using ClonTech DNA SMRT kit


  • There is ~2ul of the probe left in the tube after aliquot for single-use. I added 3ul of H2O to increase the volume to 5ul. I then took 1ul for QUbit ssDNA quantification.
  • Qubit result = 5.85ng/ul --> original concentration from Nimblegen should be ~ 14.62ng/ul.
  • I use 10ng or 1.71ul for DNA SMART ChIP seq experiment. This amount is the maximum amount recommended by the kit (between 100pg-10ng of dsDNA or ssDNA)



A: Denaturation of DNA

- I set up reaction by including bisDNA using BisulFlash DNA bisulfate Conversion kit from Epigentek performed on 141028 to test contamination issue of using bead based kit.
No. Template Input amount BIS-CVT Stock conc. Volume (ul) Dilution buffer Total (ul)
#1 Control fragmented DNA 100pg YES NA 20.00 0.00 20.00
#2 Control fragmented DNA 1ng YES NA 20.00 0.00 20.00
#3 Control fragmented DNA 10ng YES NA 20.00 0.00 20.00
#4 NTC NA YES NA 20.00 0.00 20.00
#5 EZ Epi hg19 UMR v1 10ng no 5.85 1.71 18.29 20.00
#6 Control fragmented DNA 1ng no 0.5 ng/ul 2.00 18.00 20.00
#7 NTC NA no NA 0.00 20.00 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 7.2 rxn Dephosphorylation Master Mix for all reactions by combining the following reagents on ice.
Components 1rxn (ul) 7.2 rxn
DNA SMART Buffer 3.25 23.4
Shrimp Alkaline Phosphatase 0.75 5.4
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) 7.2 rxn
DNA SMART T-Tailing Mix 1.00 7.2
Terminal Deoxynucleotidyl Transferase 1.00 7.2
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