AlanFung:LabNotes/CTCF/2011-2-14
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Nextera Methyl-Seq DNA Sample Prep
Objective
- Confirmation of the library prep with beta nextera methyl-seq kit with input DNA (100ng starting amount) with GM12878 gDNA.
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
- Qiagen Purification Kit
Overview
- Tagmentation
- Purification
- Fill-in
- Purification
- Bisulfite Conversion
- Purification
- bPCR-compatible sites and library enrichment
Protocol
- A. TagmentationTM Reaction
- 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:
xul Nuclease-Free Water 2ul 100ng Target DNA 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 a Zymo DNA Clean & Concentrator-5 Kit (or equivalent). Brief Zymo Protocol (perform at room temperature): -Add 100 μl of DNA Binding Buffer to 20 μl Tagmentation Reaction. -Mix briefly by vortexing, and transfer the mixture to a Zymo-SpinTM Column in Collection Tube. -Centrifuge at 10,000 x g for 60 seconds. Discard flow-through.
-Add 250 μl of Wash Buffer to the column. Centrifuge at 10,000 x g for 60 seconds. -Discard the flow-through and repeat wash step. -Centrifuge the empty column at 10,000 x g for 60 seconds to dry and eliminate any residual Wash Buffer. -Transfer the column to a clean and sterile 1.5-ml microcentrifuge tube -Add 11 μl of Nuclease-Free Water directly to the column and incubate at room temperature for 1-2 minutes. Centrifuge at 10,000 x g for 60 seconds to elute the DNA. -The final eluted volume should be ~10 μl. 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 70oC for 10 minutes. 6. Purify the Fill-in reaction using a Zymo DNA Clean & Concentrator-5 Kit, as in Step 4 above.