AlanFung:LabNotes/CTCF/2011-8-26
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Low-input Bisulfite Conversion and MDA Nextera library
Protocols Today
- Start with four tubes: 314 cells, 157 cells x 2, NTC all go through bisulfite conversion
- Make 1571 cell/ul (original tube)
- Make 314 cells/ul (dilute 10ul cells in 40 ul ddh2o)
- Make 157cells/ul (dilute 10ul cells in 90 ul ddh2o)
Cell Lysis
- First, make protease Dilution
- Add 3 uL Protease to 97 uL H2O for a 3:100 dilution
- Next, add 1 uL 3:100 diluted Protease and 1 uL 3X Lysis Buffer (NP40) to 1 uL Frozen cells; incubate at 37 C for 30 minutes, followed by 70 C for 20 minutes
- Cells are now lysed
Make New BSA-Water solution
- Stock solution is at 10mg/ml, need to dilute to 0.5mg/ml
- Take 10ul stock BSA dilute with 190ul nuclease free water
Bisulfite Conversion - Imprint Kit
- Prepare DNA Modification Solution (CT Conversion Reagent):
- Add 21 uL 0.5 mg/mL BSA and
- Add 1 uL Imprint Balance solution to each sample;
- incubate at 37 C for 10 minutes
- Add 125 uL DNA Modification solution to each tube, incubate at 65 C for 90 minutes
- Perform Column Purification of each sample:
- Elute into 11uL Elution Buffer
Continue on 8/28
- Denature all samples at 95C for 30sec and quickly transfer to cooler rack
Content | Volume | MM |
10X Repli Phi Buffer | 2 | 8.8 |
1mM N9 Primer | 1 | 4.4 |
25mM dNTP | 0.8 | 3.52 |
Repliphi Phi 29 (100U/ul) | 1 | 4.4 |
Klenow Exo Minus (10U/ul) | 0.5 | 2.2 |
Nuclease free H20 | 4.7 | 20.68 |
Modified DNA Sample | 10 | Do not add to MM |
Total | 20 | 44 |
- Add 10ul of MDA mastermix to each sample
- 30C 60 min, 65C 10 min, 4C hold
MinElute
- Elute with 10ul nuclease free water
Speedvac
- To get 3ul for shendure's method of nextera library tagmentation, I will transfer the 10ul samples to qpcr tube, put the cap on and puncture a hole at the cap and speedvac till the sample is dry
- took about 28min to dry samples
- Re-dissolve the samples with 3ul of nuclease free water, sit at RT for 5 min
Tagmentation
- To lysate, add 1 uL 1:50 diluted Nextera Transposase and 1 uL 5x Nextera HMW Buffer; incubate at 55 C for 5 minutes
- To make 1:100 Protease, mix 1ul of protease with 99ul of nuclease free water
- Next, add 1 uL 1:100 diluted Protease to solution; incubate at 37 C for 30 minutes, followed by 70 C for 20 minutes
- Prepare mastermix for library amplification
Content | Volume | MM |
Nuclease Free H2O | 16 | 70.4 |
10uM Orange Primer | 1 | 4.4 |
10uM Blue Primer | 1 | 4.4 |
Tagmented DNA | 6 | Do not add to MM |
2X Kapa SYBR Supermix | 25 | 110 |
BST | 1 | 4.4 |
Total | 50 | 193.6 |
- Add 44ul MM to each reaction
- Perform qPCR using the following reaction conditions:
- 65 C, 3 min incubation (BST gap filling)
- 95 C, 30 seconds (Denature)
- 35 cycles of:
- 95 C, 10 seconds (Denature)
- 58 C, 30 seconds (Anneal)
- 72 C, 3 minutes (Extension)
- 72 C, 5 minutes (final extension)
- Hold at 4 C
- 300 cells was taken out after 7 cycles
- 150 cells & NTC was taken out after 12 cycles
File:ZhangLab 2 2011-08-28.jpg
Purification
- Minelute column, elute with 15ul water
- Nanodrop gave me weird results, I was having problem when measuring the blank sterile water. Will avoid using the water provided by the Repliphi phi 29 kit for elution next time
- 150:6.6ng/ul
- 300:-0.2ng/ul
- Use Qubit to measure the concentration instead
- 150:4.16ng/ul
- 300:1.46ng/ul
- Looks like I do not have enough samples for end repair, may be I should run a few more cycles of PCR?
- I would like to run a gel to confirm the size of the amplicons
Continue on 8/29
- Talk to Dr. Zhang and he suggests that I can take 0.5ul of amplified samples and repeat the PCR, doing that will avoid over amplify all the samples.
- Also me and Athurva need to come up with a plan on experiments to obtain a few aspects that we need to validate
- whether bisulfite conversion is complete
- whether the methylation level is accurately capture in the libraries
- whether the resulting amplicons contain the right adaptors such that they can be sequenced on the illumina platform
- whether all genomic regions are present at roughly equal abundance in the libraries
- What can you get from the Sanger sequencing experiment, and what other experiments you need to do
Library Amplification
Content | Volume | MM |
Nuclease Free H2O | 22.5 | 74.25 |
10uM Orange Primer | 1 | 3.3 |
10uM Blue Primer | 1 | 3.3 |
Purified Amplicons | 0.5 | Do not add to MM |
2X Kapa SYBR Supermix | 25 | 82.5 |
Total | 50 | 165 |
- Add 49.5ul MM to each reaction
- Perform qPCR using the following reaction conditions:
- 95 C, 30 seconds (Denature)
- 35 cycles of:
- 95 C, 10 seconds (Denature)
- 58 C, 30 seconds (Anneal)
- 72 C, 3 minutes (Extension)
- 72 C, 5 minutes (final extension)
- Hold at 4 C
Minelute
- Minelute with 15ul EB buffer
- Nanodrop Result
- NTC:25.9ng/ul
- 150:94.1ng/ul
- 300:85.5ng/ul
PAGE Gel Analysis
File:ZhangLab 2 2011-08-29 18hr 51min.jpg
Continue on 8/30
PAGE Size Selection
- Talked to Noi, she suggests that I can load ~500ng/well for my sample size is a a big range, I will load 2 wells per sample
- Load a 5well TBE gel into the gel running module
- Add in fresh 0.5X TBE buffer to the inside core full the outer core up to the metal line
- Dilute sample to no more than 40ng/uL
Mix 1ul low mass ladder with 10ul dH2O, 9ul 6X loading dye Mix 6.5ul samples with 4.5ul dH2O, 9 ul 6X loading dye
- Load the samples into the wells, run at 200V for 30 min.
- Stain gel with 4uL SYBR Gold in ~100 mL of TBE in clean tray.
- Place tray on an orbital shaker for ~5 min.
Purify the product from the gel
- Prepare two gel shearing assemblies by placing a 0.5 mL tube within another 1.5 mL Non-Stick tube. Use G20 needle to punch a small hole at the bottom of the 0.5 mL tube. Use two assemblies for each PAGE gel.
- Cut out the correct band (175-800 nt in this case), chop the band into small slices, split into two half and transfer each half to one shearing assembly.
- Centrifuge at top speed > 12000 rpm for 2 min to sheer the gel, remove the 0.5 mL tube.
- Add 400 uL clean 1x TE buffer in the 1.5 mL tube. Tape the tubes on a vortexer, place the vortexer in 37C incubator, shake the tubes at low speed for 1 hour. Alternatively, can keep the tubes in 4C fridge overnight.
- Spin down all tubes at 13,000rpm for 2min
- In the PCR hood, transfer the liquid layer of the gel mix to Nanosep columns, spin for 3 min at top speed (15,000 rpm), and then transfer supernatant to 1.5mL tube.