AlanFung:LabNotes/CTCF/2011-4-25: Difference between revisions
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==Objective== | ==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. | *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 | *Adapt Shendure's protocol for low input library preparation | ||
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*Increase yield from the bisulfite conversion using zymo methylation direct kit (works with 50pg starting amount) | *Increase yield from the bisulfite conversion using zymo methylation direct kit (works with 50pg starting amount) | ||
*Test Kapa Hifi polymerase | *Test Kapa Hifi polymerase | ||
==Samples & Materials== | ==Samples & Materials== | ||
*Nextera Sample Prep Kit_Epicentre_NMS072910B | *Nextera Sample Prep Kit_Epicentre_NMS072910B |
Revision as of 00:34, 26 April 2011
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)