AlanFung:LabNotes/CTCF/2011-4-25

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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]

  • Previous experiment was carried out using methylation gold kit, which doesn't work as well as the direct kit, this this we will take the advice from zymo tech support and modify the protocol of the methylation direct kit
  • 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
  • Improve the yield from the bisulfite conversion step using suggestions made by zymo tech support
  • Increase yield from the bisulfite conversion using zymo methylation direct kit (works with 50pg starting amount)

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 (Methylation Direct Kit)

Overview[edit]

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

Protocol[edit]

TagmentationTM Reaction[edit]

  • Dilute GM12878 gDNA 1:100 to 176ng/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:
3.41ul   600pg NA12878 gDNA (176pg/ul qubit check on 03/31/11)
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)
5.4ul        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 9.09ul ddh2o make the total volume up to 20ul
  • 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 14,000rpm for 30s
  • Discard flow through
  • Add 200uL of M-Desulphonation Buffer to column let stand at RT for 14m
  • 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 warm M-Elution Buffer (37C) directly to the column matix, sit at 37C for 5 min
  • Repeat Centrifuge

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

  • Cut the volume in half
  • 1. Assemble the following reaction components at room temperature
10ul bilsufite converted library
12.5ul 2X Nextera PCR Buffer
0.5ul 50X Nextera Primer Cocktail 
0.5ul 50X Nextera Adaptor 2
0.5ul Nextera MEthyl-Seq Polymerase
0.5ul Sybr Green
  • 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
72C hold for 1min
15C forever
  • 3. Purify the tagged DNA using qiaquick column
  • Page Quantification

File:ZhangLab 2 2011-04-27 14hr 18min crop.jpg

Results[edit]

  • Based on the page quantification, the library construction is successful, although we are having primer dimer issues and a huge smear, we do see brighter bands at 200-400 bp
  • Will move on to do cloning and Sanger Sequencing results