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===TagmentationTM Reaction === *Dilute GM12878 gDNA 1:100 to 429pg/ul *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: 13.60ul Nuclease-Free Water 1.40ul 600pg NA12878 gDNA (429pg/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. Shendure protocol for reference Low input transposase-based library preparation For the 500 pg and 100 pg E. coli (CC118) libraries and 10 pg human library (NA18507, Coriell), genomic DNA (in 1 μl volume) was incubated with 1 μl Nextera Illumina-compatible transposomes (Epicentre) at a 1 to 50 dilution (1 μl Nextera enzyme, 24 μl TE, 25 μl 100% glycerol), 1 μl 5× Nextera HMW buffer, and 2 μl nuclease-free water (Ambion). To avoid contamination, all dilutions and reaction preparation was carried out in a PCR hood. Reactions were incubated at 55°C for 5 min followed by addition of 25 μl 2× Nextera PCR buffer, 0.5 μl SYBR Green, 1 μl 50× Nextera primer cocktail, and 1 μl 0.5 μM barcode adaptor 2 (barcodes A6, A9, or A4 for 500 pg and 100 pg E. coli DNA, or 10 pg human DNA, respectively) and cycled under standard Nextera conditions in a MiniOpticon (Bio-Rad) real-time PCR thermocycler. Both reactions were removed after 20 cycles and cleaned up using Qiaquick MinElute columns Eluting in 20 μl EB. Libraries were run on a 6% Novex TBE PAGE gel (Invitrogen) for size verification and Sequenced as barcoded spike-ins as per standard Illumina GAIIx protocol as a paired-end 101 bp (plus 9 bp barcode) run for E. coli libraries and a paired-end 36 bp (plus 9 bp barcode) run for human.
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