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
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 250V for 25 min.
- Stain gel with 2.5uL 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.
File:ZhangLab 2 2011-08-30 15hr 04min.jpg
- Centrifuge at top speed > 13000 rcf 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
- Spin down all tubes at 13,000rcf 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.
Precipitation
- Turn on centrifuge let it cool down to 4C
- Add 0.1x total volume of 3M sodium acetate and 2.5x total volume 100% ethanol. (35ul 3M NAoAC, 875ul 100% ETOH)
- Add 1uL Glycoblue vortex and spin down so that one can precipitate better and see the DNA.
- Freeze in -80C for 20 min, spin at 10,000 rpm for 20 min at 4C, discard supernatant with pipette.
- Add 500 uL of 75% ethanol to wash DNA, spin at 10,000 rpm for 5 min at 4C, discard supernatant and aspirate with (P1000 pipette) let dry for 5 mins spindown ad aspirate with P200 pipette.
- Let dry in hood ~ 10 min.
- Add 5 uL of water to each tube to resolve the DNA , leave at least 15 min.
- Combine all 4 tubes into one
Qubit Measurement
- 150cells:6ng/ul
- 300cells:10.7ng/ul
Cloning for Sequencing
Blunting with NEB end repair kit
- Take 8.5ul of PCR product
Content | 150 | 300 |
Fragmented DNA | 8.5 | 8.5 |
10X End repair reaction buffer | 1 | 1 |
End reapir enzyme | 0.5 | 0.5 |
H2O | 0 | 0 |
Total Volume | 10 | 10 |
- Incubate at 20C for 30min
- Minelute with 10ul EB
Qubit
- 150cells:2.84ng/ul
- 300cells:5.12ng/ul
Ligating into pCR blunt vector
- Calculate the amount of PCR product needed for ligation
- x ng insert =(10*Y bp pcr product*25ng linearized pcr blunt)/3500bp pcr blunt
- Size of library is about 500bp
- 35.71ng needed, will use 1ul for both samples
- Incubate at 16C for 1 hr, hold at 4C continue after dinner