AlanFung:LabNotes/CTCF/2011-3-29

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Nextera Methyl-Seq DNA Sample Prep on 100cells (600pg DNA)[edit]

Low input transposase-based library preparation (Shendure's Protocol)[edit]

Objective[edit]

  • Confirmation of the library prep with beta nextera methyl-seq kit with input DNA (100 cells, ~600pg DNA starting amount) with GM12878 gDNA.
  • Adapt Shendure's protocol for low input library preparation
  • Perform bis-cut with carrier and replace column purification step during bis-cut with ethanol precipitation


Samples & Materials[edit]

  • Nextera Sample Prep Kit_Epicentre_NMS072910B
NexteraTM Methyl-Seq Tagmentation Mix (Illumina-compatible) 
5X Nextera Reaction Buffer (HMW)
50X Nextera Primer Cocktail (Illumina-compatible) 
50X Nextera Adaptor 2 (Illumina-compatible) 
2X Nextera Fill-in Buffer 
2X Nextera PCR Buffer 
200X Nextera Read 1 Primer 
200X Nextera Read 2 Primer 
200X Nextera Index Read Primer  
Nextera Methyl-Seq Polymerase 
  • Input DNA
  • Bisulfite conversion kit
  • Qiagen Purification Kit

Overview[edit]

  • Tagmentation with diluted transposon at low volume (5ul)
  • Fill-in
  • Purification
  • Bisulfite Conversion with lambda phage DNA
  • Purification
  • bPCR-compatible sites and library enrichment

Protocol[edit]

TagmentationTM Reaction[edit]

  • Dilute GM12878 gDNA 1:100 to 429pg/ul
  • 1. Prior to assembling the reaction, briefly centrifuge ALL tubes to assure that the reagents are at the bottom of the tubes.
  • 2. Assemble the following reaction components on ice, in the order listed:
1.6ul     ddH2O
1.40ul   600pg NA12878 gDNA (429pg/ul nanodrop check on 2/15/11)
1ul         5X Nextera HMW Reaction Buffer
1ul         1:50 Nextera Methyl-Seq Tagmentation Mix (Illumina-compatible) ((1 μl Nextera enzyme, 24 μl TE, 25 μl 100% gly- cerol)
5ul        Total reaction Volume
  • 3. Mix briefly by pipetting, and incubate at 55C for 5 minutes.
  • 4. Add 5 μl of 2X Nextera Fill-in Buffer and 0.5 μl of Nextera Methyl-Seq Polymerase to 5 μl of the Tagmented DNA.
Mix and incubate at 70C for 10 minutes.

Bisulfite Conversion of Tagged Nextera Fragments[edit]

  • Add in 130uL of CT conversion Reagent Solution directly to the digested samples
  • Vortex and spin down
  • Perform reaction in thermocycler
      Step1   98C, 8m
      Step2   64C, 3.5hr
      Step4   4C,  storage for up to 20 hr
  • Add 600uL of M-Bindin Buffer into a IC Column
  • Load 150uL of samples into IC column

CLOSE CAP AND MIX BY INVERTING THE COLUMN SEVERAL TIMES

  • Centrifuge at 20,000g for 30s
  • Discard flow through
  • Add 100uL M-Wash Buffer to column
  • Repeat Centrifuge
  • Add 200uL of M-Desulphonation Buffer to column let stand at RT for 20m
  • Repeat centrifuge step
  • Add 200uL of M-Wash Buffer to the column
  • Repeat Centrifuge
  • Place column in a 1.5mL tube
  • Add in 11uL of M-Elution Buffer directly to the column matix, sit at RT for 5 min
  • Repeat Centrifuge

Addition of bPCR-Compatible Sites and Library Enrichment[edit]

  • 1. Assemble the following reaction components at room temperature
10ul bilsufite converted library
25ul 2X Nextera PCR Buffer
1ul 50X Nextera Primer Cocktail 
1ul 50X Nextera Adaptor 2
1ul Nextera MEthyl-Seq Polymerase
  • 2. Cycle the samples in a thermocycler under the following conditions
95C for 30sec
25cycles of
95C for 10sec
60C for 10sec
72C for 1min
hold for 1min

File:3 30 2011.JPG

  • 3. Purify the tagged DNA using qiaquick column
  • Page Quantification

File:2011-03-30 NTC.jpg

Conclusion[edit]

  • Amplification is successful, yet page quantification reviews that the band occurs around 150bp, which is not that right size of the library. According to expicentre, the correct size of the library should be ~175-500bp. Previous experiment with 100ng starting amount of gDNA show bands around 200 to 400bp. I think the band is most likely primer dimers. However when you view carefully at the gel image you will see a light smear of dna, that could be the library. Now if we can further enrich the yield of the library, we can possibly get a good amplicon out of it. I contacted Zymo's tech suport and Zeith was able to help me trouble shoot the problem, and he suggested a few improvements that can be made and could theoretically increase the yield.
  • Make new wash buffer
  • The washing step after binding can be omitted
  • Perform desulphonation step no longer than 15mins
  • warm the elution buffer up to 37C before use
  • Always use new CT conversion reagent