AlanFung:LabNotes/CTCF/2011-5-16: Difference between revisions

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Line 102: Line 102:
  0.5 uL 0.5uM Short Orange  
  0.5 uL 0.5uM Short Orange  
  0.5 uL 0.5uM Short Blue
  0.5 uL 0.5uM Short Blue
  10 uL DNA/Water
  10 uL DNA
  1 uL Klenow
  1 uL Klenow
  25 uL KAPA Supermix  
  25 uL KAPA Supermix  

Revision as of 17:47, 17 May 2011

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

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

Objective

  • 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 by Klenow DNA polymerase prepared by Arthurva 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

  • 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

  • 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

TagmentationTM Reaction

  • 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
  • 3. Mix briefly by pipetting, and incubate at 55C for 5 minutes.
  • Add 1 uL Klenow Polymerase and 1 uL methyl-Biotin-dNTP Mix
  • Incubate for 40min at 30 C

Bisulfite Conversion of Tagged Nextera Fragments

    • Add 13ul ddh2o to all tubes make it 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

Dilution of 50ng positive control

  • ssDNA qubit quantification
    • Results

2nd Strand Synthesis and Library Enrichment

  • 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
0.5ul 0.5uM Orange Primer
0.5ul 0.5uM Blue Primer
    • Perform Manufacturer protocol for KAPA
    • Cycle the samples in a thermocycler under the following conditions
  • 3. Purify the tagged DNA using qiaquick column
  • Page Quantification

Results