AlanFung:LabNotes/CTCF/2011-2-14: Difference between revisions

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*Library successfully amplified with the adapters
*Library successfully amplified with the adapters
*Band shows up around 200BP which is expected, as the transposon shears the DNA to around that size. It appears that the Meth-Seq DNA sample prep kit is working
*Band shows up around 200BP which is expected, as the transposon shears the DNA to around that size. It appears that the Meth-Seq DNA sample prep kit is working
*Library preparation with Methy-Seq DNA Prep kit is successful with 100ng of starting material


==Suggestions==
==Suggestions==

Latest revision as of 18:19, 18 February 2011

Nextera Methyl-Seq DNA Sample Prep[edit]

Objective[edit]

  • Confirmation of the library prep with beta nextera methyl-seq kit with input DNA (100ng starting amount) with GM12878 gDNA.

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
  • Purification
  • bPCR-compatible sites and library enrichment

Protocol[edit]

TagmentationTM Reaction[edit]

  • 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:
12.67ul Nuclease-Free Water 
2.33ul   100ng NA12878 gDNA (42.9ng/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.

Bisulfite Conversion of Tagged Nextera Fragments[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
  • 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. Add another 200ul of M-wash buffer and centrifuge for an additional 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

Results[edit]

File:ZhangLab 2 2011-02-17 15hr 18min.jpg

  • Library successfully amplified with the adapters
  • Band shows up around 200BP which is expected, as the transposon shears the DNA to around that size. It appears that the Meth-Seq DNA sample prep kit is working
  • Library preparation with Methy-Seq DNA Prep kit is successful with 100ng of starting material

Suggestions[edit]