Noi/NOTES/2014-10-29
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Generation of sequencing library of a customized SeqCap EZ Epi Probe Pool to decode the sequences[edit]
- 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 (actual volume is more than 54ul)
Things to do upon receive the probe pool[edit]
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 to decode the sequences of the probe pool
- Take a small aliquot of the left over probe pool after a single-use aliquots of 16 rxn for library prep using ClonTech DNA SMRT ChIP-Seq 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 of a diluted probe 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[edit]
- - 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[edit]
- - 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[edit]
- - 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
D: Primer Annealing and Template Switching Reaction[edit]
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 7.2rxn Template Switching Master Mix for all reactions by combining the following reagents on ice.
Components 1rxn (ul) 7.2 rxn DNA SMART Buffer 6.00 43.20 DNA SMART Oligonucleotide mix 6.00 43.20 SMARTScribe Reverse Transcriptase 4.00 28.80 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[edit]
- I would initially do a quick qPCR to test the PCR cycle number
Components | Total 100ul | Total 15ul | 8.5 rxn |
SeqAmp PCR Buffer (2X) | 50.00 | 7.50 | 63.75 |
Forward PCR Primer (12.5uM) | 2.00 | 0.30 | 2.55 |
Reverse PCR Primer (12.5uM, R3) | 2.00 | 0.30 | 2.55 |
50X SYBR | 0.40 | 0.06 | 0.51 |
SeqAmp DNA Polymerase | 2.00 | 0.30 | 2.55 |
Total | 56.40 | 8.46 |
- - Aliquot 8.46ul + 6.6ul of DNA template
- - Split 4.5ul to 3X 8-tube strip
- - ChIP-PCR: 94C, 1min -->[98C, 15s -> 55C, 15s, 68C, 30s] 11X only for Epi hg19 UMR, 15X, 16X, 18X --> 4C hold
Note: I used REalplex (Eppendorf) for amplification
- - Load all 4.5ul PCR product in 6% TBE gel.
- Note: To save the TBE gel, I did not load all PCR cycles. Since 10ng of Hg19 LMS v1 shoot up very quick at 11 cycle as recommended by the kit, I stopped it earlier.
File:ZhangLab 2 2014-10-29 17hr 52min DNASMRT-11XEpi-16x.jpg * : over-amplified ** I made a wrong labeling. It should be UMR instead of LMS File:ZhangLab 2 2014-10-29 17hr 52min DNASMRT-18x.jpg
- From PAGE verification, I saw the PCR product size of Hg19 LMS v1 between ~180-almost 300bp. I really had no idea what is the actual size of sequencing library should be as I did not know the actual length of the probe. I believed that the experiment worked since I did not see this signal in NTC reaction. I continued to amplify the left over template (37.4ul) for 12 cycles.
Components | Total 100ul | Total 85ul |
SeqAmp PCR Buffer (2X) | 50.00 | 42.50 |
Forward PCR Primer (12.5uM) | 2.00 | 1.70 |
Reverse PCR Primer (12.5uM, R10) | 2.00 | 1.70 |
50X SYBR | 0.40 | 0.34 |
SeqAmp DNA Polymerase | 2.00 | 1.70 |
Total | 56.40 | 47.94 |
- - ChIP-PCR: 94C, 1min -->[98C, 15s -> 55C, 15s, 68C, 30s] X12 --> 4C hold
- - To save all product above 100bp, I use a little high ratio of AMPure bead (1.8X) to purify the PCR product and eluted with 30ul H2O
- - I took 4ul for PAGE verification. I also included 2ul of diluted original probes from Nimblegen (total amount ~11.7ng) to verify the size in 6% TBU gel
File:ZhangLab 2 2014-10-29 20hr 59min TBU-verify Epi-LMS-v1-ori-seqlib.jpg * I made a wrong labeling. It should be UMR instead of LMS
- It look like I still have some background of the band around 150bp and primer dimers. To may need to do one more round of bead purification to exclude these background.
2014-11-07[edit]
- I added more ~25ul H2O to bring to volume of the DNA SMRT Seq probe pool UMR to 50ul and re-purified with 1X AMPure bead to remove the lower bands.
- I resuspend the library with 25ul EB buffer
- Verify purified library in 6% TBE gel
File:ZhangLab 2 2014-11-07 18hr 48min beadpurified UMRv1-SeqCap-Normal-cancer-3ul.jpg Library ID: 1 = NP-DNASMRT-Dec_SeqCapUMRv1-Oct29 2 = NP-SeqCap-pNormalSetA-Nov4 3 = NP-SeqCap-pCancerSetA-Nov4
- Library ID: NP-DNASMRT-Dec_SeqCapUMRv1-Oct29, 2014
- Average size: 180-300bp
- Primer system: Illumina index
- Index:10, Seq(RC) = TAGCTT
- I will hand library to Derek for qPCR quantification and sequencing in the future.