AlanFung:LabNotes/CTCF/2011-2-28

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Nextera Methyl-Seq DNA Sample Prep on 100cells (600pg DNA)[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. The challenge of this experiment will be the sample lost due to purification steps and bisulfite conversion step. Qiagen claims 90% recovery rate for the purification columns while Zymo claims a 75% recovery rate for the bisulfite conversion. Resulting in ~360pg of sample library before the library enrichment. Since the recovery rate for the bisulfite conversion is low we will be adding lambda phage dna to the sample to "share" the lost and hopefully will minimize the lose of sample.

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
  • Purification
  • 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:
13.60ul Nuclease-Free Water 
1.40ul   600pg NA12878 gDNA (429pg/ul nanodrop check on 2/15/11)
4ul         5X Nextera HMW Reaction Buffer 
1ul         Nextera Methyl-Seq Tagmentation Mix (Illumina-compatible)
20ul       Total reaction volume 
  • 3. Mix briefly by vortexing, and incubate at 55C for 5 minutes.
  • 4. Purify the tagmented DNA using Qigen column and elute with 11ul Elute buffer
  • 5. Add 10 μl of 2X Nextera Fill-in Buffer and 1 μl of Nextera Methyl-Seq Polymerase to 10 μl of the eluted/Tagmented DNA.
Mix and incubate at 70C for 10 minutes. 
  • 6. Purify the Fill-in reaction using a Qiagen column as in Step 4 above.
Shendure protocol for reference
Low input transposase-based library preparation
For the 500 pg and 100 pg E. coli (CC118) libraries and 10 pg human library (NA18507, Coriell), genomic DNA (in 1 μl volume) was 
incubated with 1 μl Nextera Illumina-compatible transposomes (Epicentre) at a 1 to 50 dilution (1 μl Nextera enzyme, 24 μl TE, 25 μl 100% glycerol), 
1 μl 5× Nextera HMW buffer, and 
2 μl nuclease-free water (Ambion). 
To avoid contamination, all dilutions and reaction preparation was carried out in a PCR hood. 
Reactions were incubated at 55°C for 5 min 
followed by addition of 25 μl 2× Nextera PCR buffer, 0.5 μl SYBR Green, 1 μl 50× Nextera primer cocktail, and 1 μl 0.5 μM barcode adaptor 2 (barcodes A6, A9, or A4 for 500 pg and 100 pg E. coli       DNA, or 10 pg human DNA, respectively) and cycled under standard Nextera conditions in a MiniOpticon (Bio-Rad) real-time PCR thermocycler. 
Both reactions were removed after 20 cycles and cleaned up using Qiaquick MinElute columns
Eluting in 20 μl EB. 
Libraries were run on a 6% Novex TBE PAGE gel (Invitrogen) for size verification and 
Sequenced as barcoded spike-ins as per standard Illumina GAIIx protocol as a paired-end 101 bp (plus 9 bp barcode) run for E. coli libraries and a paired-end 36 bp (plus 9 bp barcode) run  for human.

Bisulfite Conversion of Tagged Nextera Fragments with carrier[edit]

  • 1. Add 900ul water, 300ul of M-Dilution Buffer, and 50ul M-Dissolving buffer to a tube of CT Conversion Reagent
  • 2. Mix at room temperature with frequent vortexing or shaking for 10 minutes
  • 3. Preparation of M-Wash buffer
  • 4. Mix 10ul nuclease free water with 130ul of the CT Conversion Reagent to 10ul of your DNA sample in a PCR tube
  • 5. Place the sample tube in a thermal cycler and perform the following steps
98C for 10mins
64C for 2.5hrs
4C forever
  • Add in 500ng yeast tRNA carrier
  • 6. Add 600ul M-binding buffer to a Zymo spin column and place the column into a provided collection tube
  • 7. Load the sample into the column. Close the cap and mix by inverting the column several times
  • 8. Centrifuge at full speed for 30sec. Discard the flow through
  • 9. Add 100ul M-wash buffer to the column. Centrifuge at full speed for 30sec
  • 10. Add 200ul M-Desulphonation Buffer to the column and let stand at room temperature for 20mins. After incubation centrifuge at full speed for 30sec
  • 11. Add 200ul M-Wash buffer to the column. Centrfuge at full speed for 30sec.
  • 12. Place the column into a 1.5ml microcentrifuge tube . Add 15ul of M-Elution Buffer directly to the column matrix. Centrifuge for 30sec at full speed to elute the DNA

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

  • 1. Assemble the following reaction components at room temperature
12ul Nuclease-Free water
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
  • 3. Purify the tagged DNA using qiaquick column
  • Page Quantification

File:ZhangLab 2 2011-03-10 16hr 01min.jpg

Results[edit]

  • Amplification curve shows up on RT-PCR
  • No bands showed up on the PAGE quantification
  • I suspect the samples are lost in the purification step

Improvements[edit]