AlanFung:LabNotes/CTCF/2011-10-4

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Notes[edit]

  • From previous experiment, it is verified that cells can be digested using NaOH. I will repeat experiment with the following improvements.
    • NaOH cell digestion, avoid using NP40 and protease, save time and effort.
    • EtOH precipitation after MDA, prevent size elimination with column purification and avoid spending time to speedvac to cut down the sample volume for tagmentation

Protocols Today[edit]

  • 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 & Bisulfite Conversion[edit]

150cells & Negative control[edit]

  • Add 23ul 0.5 mg/mL BSA
  • Add 1ul imprint Balance solution to each sample (150 GM12878cells/ul & ddH2O)
  • 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

MDA[edit]

  • Denature all samples at 95C for 30sec and quickly transfer to cooler rack
Content Volume MM
10X Repli Phi Buffer 2 6.2
1mM N9 Buffer 1 3.1
25mM dNTP 0.8 2.48
Repliphi Phi 29 (100U/ul) 1 3.1
Klenow Exo Minus (10U/ul) 0.5 1.55
Nuclease free H2O 4.7 14.57
Modified DNA Sample 10 Do not add to MM
Total 20 31
  • Add 10ul of MDA mastermix to each sample
  • 30C 60 min, 65C 10 min, 4C hold

EtOH Precipitation[edit]

  • Elute with 3ul nuclease free water
  • Incubate at RT for at least 10min
  • Transfer samples to qPCR strip tubes for tagmentation and qPCR

Tagmentation[edit]

  • 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)
    • 20 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
  • Stopped reaction at cycle 17
  • Purify with minelute, elute with 15ul of EB buffer, keep 1ul for gel

Bisulfite Verification w CHR8,21&22[edit]

MM CHR8 CHR21 CHR22
2X iQ Super Mix 20 20 20
Primer F (3.3uM) 6 6 6
Primer R (3.3uM) 6 6 6
gDNA 2 2 2
H2O 6 6 6
Total 40 40 40
    Step1   96C, 3m
    Step2   95C, 30s
    Step3   62C, 1m
    Step4   72C, 1m
    Step5   Go to step2 repeat 39 times
    Step6   72C, 5m
    Step7   4C,  Forever

Adding Purple and Pink Nextera Adapters[edit]

  • Using barcode 8 here
Content Volume Per Reaction
DNA Do not add to MM
Kapa HF Mix 55
Adaptor 1 2.2
Adapor 2 (Barcode) 2.2
H2O 17.6
Total 77
  • Added 0.4ul 50X SYBR Green to each reaction
Nextera_2nd
95C 30sec
[95C 10sec; 60C 30sec; 72C 2min] X 2
[95C 10sec; 62C 30sec; 72C 2min] X 3
72C, 3min

Page Gel Analysis[edit]

  • I will run a gel with the CHR8,21,22 samples and the blue/orange primers amplified samples w. -ve control

Results[edit]

  • The bisulfite conversion pcr test failed for some reason. Mostly due to old sodium bisulfite reagent. It was stored at -20C as recommended, but it is better to work with freshly prepared bisulfite conversion reagents. Will spend more time to investigate on this if possible.
  • The 1st amplification worked out really nice, with the exact amplicon size as expected and negative control didn't get amplified.
  • However after the 2nd amplification, unspecific bands showed up for both the sample and the control. I suspect this is due to lost of samples due to purification using the column.
  • I would like to try Rui's protocol where she did purification with beads and she had pretty good results from it.