AlanFung:LabNotes/CTCF/2011-7-12

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Improved protocol for Identifying source of loss during Nextera Bisulfite conversion with 1ng gDNA[edit]

Objective[edit]

  1. Want to figure out the source of loss during Nextera Bisulfite conversion
  2. Use qPCR to identify the source of the loss
  3. Eliminate source of contamination

Samples[edit]

Protocol 1 2 3 4 5 6 7
Low-Input Nextera X X X X X X X
Protease Digestion (1:100 Dilution) X X X X X X X
Klenow Extension X X X X X X X
Heat X X X
Heat w bisulfite X X
Desulphonation X X X
Column X X X X
Amplify X X X X X X X
  1. Nextera Low-Input, Normal Shendure Protocol
  2. Nextera Low-Input, Heat
  3. Nextera Low-Input, Heat, Column Purification
  4. Nextera Low-Input, Heat, Desulphonate, and Column Purify
  5. Nextera Low-Input, Heat, Bisulfite, Desulphonate, and Column Purify
  6. NTC1 - normal shendure protocol
  7. NTC2 -shendure + bisulfite protocol
  • "Nextera Low-Input": Perform Shendure Protocol, Protease Treat with 1/100 Dilution, Klenow Extend
    • Used 1 uL of 1 ng/uL Jurkat DNA in first five tubes, 1 uL H2O in NTC
  • Normal Protocol: Amplify using KapaKlenow Protocol
  • Heat: After heat, amplify using KapaKlenow Protocol
  • Heat/Column: After heat, purify through Zymo column, skipping desulphonation step; amplify using KapaKlenow Protocol
  • Heat/Desulphonate/Column: After heat, purify through Zymo column with desulphonation step; amplify using KapaKlenow Protocol
  • Heat/Bisulfite/Desulphonate/Column: After heating in bisulfite solution, purify through Zymo column with desulphonation step; amplify using KapaKlenow protocol
  • NTC1: Just do normal protocol, but with water instead of 1 ng DNA
  • NTC2: repeat shendure protocol and bisulfite conversion with NTC

Protocol[edit]

Elimination of source of contamination[edit]

  • Clean with EtOH and DNAse wash hood and UV fo 15mins
  • Use newly opened tips
  • UV all tubes
  • UV RT-PCR Grade water, EB Buffer and Protease

Low-Input Tagmentation with Nextera[edit]

  • Add 1 uL of 1:50 Diluted Nextera Transposase, 1 uL Nextera 5x HMW Buffer, and 1 uL H2O to 2ul GM12878 gDNA (0.5ng/ul)
  • Sample 1-5
Content Volume MM
1:50 Nextera Transposase 1 5
5X Nextera HMW Buffer 1 5
RT-PCR Water 1 5
0.5ng/ul gDNA 2 10
Total 5 25
  • Sample 6-7
Content Volume MM
1:50 Nextera Transposase 1 5
5X Nextera HMW Buffer 1 5
RT-PCR Water 3 15
0.5ng/ul gDNA 0 0
Total 5 25
  • Incubate at 55 C for 5 minutes

Protease Digestion[edit]

  • Add 1 uL of 1:100 diluted Qiagen Protease (5 AU/mL, diluted to 0.05 AU/mL) to solution
  • Add 5ul to sample 1-5
  • Add 2 ul to sample 6-7
  • Incubate at 37 C for 30 minutes, followed by 70 C for 20 minutes

Klenow Extension[edit]

  • Add 1 uL of 10 U/uL Klenow exo-minus DNA Polymerase and 1 uL of 1 mM Nucleotide Mix (dATP, dGTP, m-dCTP, dTTP) to solution
  • Add 5ul klenow and 5ul dNTP to sample 1-5
  • Add 2 ul klenow and 2 ul dNTP to sample 6-7
  • Incubate at 30 C for 30 minutes
  • Aliquot 8ul to each tubes
  • keep sample 1 & 6 at 4C for later amplification

Bisulfite Conversion[edit]

  • Add 130 uL of CT Conversion Reagent (Zymo Bisulfite direct Kit) to sample 5 and 7
  • Incubate at 98 C for 8 min, 64 C for 3.5 hours; hold at 4 C when finished
  • Incubate sample 2, 3, and 4 at a separate block for it has less volume

Column Purification with/without Desulphonation[edit]

  • 4, 5 and 7 desulphonation sample 3 no desulphonation
  • Set sample 2 aside until amplification
  • Follow instructions in Zymo Bisulfite Direct kit for purification
  • Skip desulphonation step when necessary

Amplification with Kapa and Klenow[edit]

Content Volume MM
DNA 10 Do not add to MM
H2O 12 92.4
10uM Orange 1 7.7
10uM Blue 1 7.7
10U/ul Klenow Exo - 1 7.7
2X Kapa 25 192.5
Total 50 308
  • KapaKlenow uses Klenow for 2nd strand synthesis, Kapa for PCR
  • Add 1 uL Orange Primer, 1 uL Blue Primer, 25 uL Kapa SYBR Supermix, and 1 uL Klenow exo-minus (10 U/uL) to solution
  • Perform PCR using the following thermocycler settings:
30 C, 10 minutes
72 C,  3 minutes
95 C, 30 seconds
==
95 C, 10 seconds
58 C, 30 seconds
72 C, 3 minutes
== x 40 cycles
72 C 10 minutes
Hold at 4 C

Results[edit]

File:07 13 11 qpcr.jpg

Protocol 1 2 3 4 5 6 7
Low-Input Nextera X X X X X X X
Protease Digestion (1:100 Dilution) X X X X X X X
Klenow Extension X X X X X X X
Heat X X X
Heat w bisulfite X X
Desulphonation X X X
Column X X X X
Amplify X X X X X X X
  1. Nextera Low-Input, Normal Shendure Protocol
  2. Nextera Low-Input, Heat
  3. Nextera Low-Input, Heat, Column Purification
  4. Nextera Low-Input, Heat, Desulphonate, and Column Purify
  5. Nextera Low-Input, Heat, Bisulfite, Desulphonate, and Column Purify
  6. NTC1 - normal shendure protocol
  7. NTC2 -shendure + bisulfite protocol
  • Results show that heat and bisulfite conversion step (sulphonation or deamination?) cause the most lost during bisulfite conversion
    1. 6 NTC shoots up earlier than #5, which is weird. It was better than the last experiment where we saw NTC shoots up first. I say the extra sterilization steps cuts down the contamination but didn't completely eliminate the source.
  • About 5 cycle difference when comparing library with and without bisulfite conversion, around 30 fold difference.

To Do[edit]

  • I guess the epigentek Bisulflash DNA modification kit will be a good candidate to cut down the DNA lost during bisulfite conversion, since the conversion time is cut down to 20 minutes