AlanFung:LabNotes/CTCF/2011-8-26

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Revision as of 20:44, 30 August 2011 by >Alan6017518 (→‎Purify the product from the gel)
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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.