AlanFung:LabNotes/CTCF/2011-5-16

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Nextera Methyl-Seq DNA Sample Prep on 50ng 100pg & 600pg DNA with negative control[edit]

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

Objective[edit]

  • Compare 100pg starting material prepared by column purification to bead bind purification
  • 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
  • Increase yield from the bisulfite conversion using zymo methylation direct kit (Claim to work with 50pg starting amount)
  • Improve the yield from the bisulfite conversion step using suggestions made by zymo tech support
  • Perform fill-in with mC-DNTP by Klenow DNA polymerase since we used up the one from Nextera Methyl Seq beta one
  • Perform 2nd strand synthesis after bisulfit conversion with Klenow which helps the protecting the fragile DNA structure after bisulfite conversion
  • Include carrier in column purification during bisulfite conversion, in order to minimise DNA lost

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 with Klenow (DNTP-mC)
  • Bisulfite Conversion with Zymo EZ DNA methylation Direct Kit
  • Column Purification with carrier
  • 2nd strand synthesis with short orange/blue primer using Klenow DNA polymerase
  • Library enrichment with orange/blue primer using KAPA

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:
Tube A
0.568ul   100pg NA12878 gDNA (176pg/ul qubit check on 03/31/11)
2.432ul           ddh2O
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
Tube B
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
Tube C
3ul           ddh2O
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
Tube D
1ul         50ng NA12878 gDNA (50ng/ul)
2ul         ddh2o
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
  • Mix briefly by pipetting, and incubate at 55C for 5 minutes.
  • We have 100mM dATP, dGTP, dTTP and 10mM m5-dCTP
  • We will make 1mM m5C-DNTP
  • Mix
    • 0.4 ul 100mM dATP
    • 0.4 ul 100mM dGTP
    • 0.4 ul 100mM dTTP
    • 4 ul 10mM m5-dCTP
    • 34.8 ul ddh2o
    • 40 ul total


  • Add 1 uL Klenow Polymerase(1U/ul) and 1 uL 1mM mC-dNTP Mix
  • Incubate for 10min at 30 C

Bisulfite Conversion of Tagged Nextera Fragments[edit]

  • Add 1.172ul 50ng ssDNA to each tube as a carrier (42.68ng/ul)
  • Add 11.83ul ddh2o to all tubes make it up to 20ul
Prepare separate tube of 1ng DNA go through bisulfite conversion with 95C 5m denaturing step
5.68ul DNA with 14.32ul h2o
  • 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

Dilution of 50ng positive control[edit]

  • ssDNA qubit quantification
    • Results 390ng/ul, this doesnt make sense
  • nanodrop RNA
    • results 29.4ng/ul
  • Make dilution to 100pg and 600pg

2nd Strand Synthesis and Library Enrichment[edit]

  • Mix on ice
0.5 uL 0.5uM Short Orange 
0.5 uL 0.5uM Short Blue
10 uL DNA
1 uL Klenow
25 uL KAPA Supermix 
13 uL Water
50ul total
  • Incubate at 30C for 10 min
  • Add
1ul 10uM Orange Primer
1ul 10uM Blue Primer
    • Perform Manufacturer protocol for KAPA
95C 3m
95C 10s
58C 30s
72C 3m
plate read
Goto step 2 repeat 34
72C 10m
Hold 15C
    • Cycle the samples in a thermocycler under the following conditions
  • 3. Purify the tagged DNA using qiaquick column
  • Page Quantification

File:ZhangLab 2 2011-05-20 11hr 32min.jpg

Results[edit]

  • The 50ng starting material library failed to amplify, however it doesn't have the same primer dimer bands at the bottom of the gel image, which is abnormal, since the negative control (Lane C) has the same bands compared to other samples. Makes me wonder there might be a mistake during the experiment leading to the lost of the library.
  • We did 35 cycles of amplification but then based on the gel image we obtain very few end products
  • Will try the new polymerase PfuTurbo Cx Hotstart DNA Polymerase and see if that can improve the yield.