Noi/NOTES/2014-10-29
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Generation of sequencing library of a customized SeqCap EZ Epi Probe Pool
- 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:
- If frozen, thaw the tube of SeqCap Epi probe pool on ice.
- Vortex the SeqCap Epi probe pool for 3 seconds.
- 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.
- 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.
- 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
- 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.
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.