AlanFung:LabNotes/CTCF/2011-4-25

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Revision as of 00:34, 26 April 2011 by >Alan6017518 (→‎Objective)
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Nextera Methyl-Seq DNA Sample Prep on 100cells (600pg DNA)

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

Objective

  • 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)
  • Test Kapa Hifi polymerase

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)