AlanFung:LabNotes/CTCF/2011-2-14
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: Nuclease-Free Water Target DNA (in T10E1 Buffer [10 mM Tris-HCl (pH 7.5), 1 mM EDTA]) 5X Nextera HMW Reaction Buffer Nextera Methyl-Seq Tagmentation Mix (Illumina-compatible) Total reaction volume 3. Mix briefly by vortexing, and incubate at 55oC for 5 minutes. Notes: To prevent evaporation, the reaction should be carried out in a thermocycler with a heated lid or the reaction should be overlaid with mineral oil. Since tagmentation occurs very slowly at room temperature, we recommend assembling the components on ice and proceeding immediately to the 55oC incubation. 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.