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=Contiguity Preserving Transposition Sequencing (CPT-Seq)= [[Daniel Jacobsen|Back to Main]] '''Notes''' *Original Protocol from [http://www.nature.com/ng/journal/v46/n12/full/ng.3119.html Amini et al] *Input to this protocol should be '''high molecular weight DNA''' (as little fragmentation as possible); [[Daniel:Protocols/HiMWDNAExtract|DNA Extraction Protocol]] ==Transposon Complex Assembly== Meant to assemble 20 different transposon-Tn5 complexes; with each containing the Tn5 mosaic end sequence at its 3' end to a 5'-phosphorylated 19-bp mosaic end complementary oligonucleotide. Final stock concentration: '''100uM'''. 8 of the oligonucleotides had ends compatible with the P5 Illumina sequencing end, and the other 12 had the adaptor for the P7 side. #Mix Tn5 mosaic ends and complementary oligonucleotides in annealing buffer ##Annealing buffer: 10mM Tris-HCl, 1mM EDTA, 25 mM NaCl, pH 8.0 #Anneal using a themocycler with following parameters: ##95C for 5 min ##decrease to 25C at -0.1C/s #Transposons were then individually mixed at a 1:1 molar ratio with EZ-Tn5 at final stock concentration of 12.5 uM #Incubate at 37C for 1 hour #Assess quality using an 8% TBE gel #Generate 96 transposome complexes ##aliquot, in a 1:1 molar ratio, 8 i5 and 12 i7 transposomes into columns 1-12 and rows A-H, respectively of a 96 well plate ##Store at -20C #Working stock is 2.5 uM and can be stored at -20C ==Transposition of High MW gDNA== #Set up 96 transposition reactions on ice in low-DNA-binding PCR plate ##1 ng Hi-MW gDNA ##10 uL of 2X Nextera Tagment DNA buffer ##8 uL water #Add 2.5 pmol of each transposome complex to its respective well; mix gently by pipetting #Incubate for 10 min at 55C in a thermocycler with a heated lid #Stop transposition by adding 20 uL of 40 mM EDTA (pH 8.0) to each reaction #Incubate for 15 min at 37C #Pool 20 uL from each well into a plastic basin and gently rock for 5 min at 2 r.p.m. to mix well #Dilute 1:25 (25 pg/uL -> 1 pg/uL) in 1X TE Buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0) #Add 10 pg of the diluted pool to each well of a low-DNA-binding 96 well PCR plate containing 10 uL of 5 uM Nextera i7 primer and 200 ng BSA #To dissociate Tn5, add 2 uL of 1% SDS and gently mix by pipetting #Incubate at 55C for 15 min ==PCR Indexing== Similar to transposon-level indexing, PCR level indexing was also performed with 8 i5 and 12 i7 PCR primers #Add 10 uL of 5 uM i5 primer to its respective well after SDS treatment #Aliquot 30 uL of Nextera PCR Master mix and water into each well from a master mix to create a total PCR volume of 100 uL #For PCR ##72C for 3 min ##98C for 30 sec ##20 cycles ###98C for 30 sec ###63C for 30 sec ###72C for 3 min #Pool all 96 PCRs and purify ##50 uL aliquots pooled ##Assess quality with High-Sensitivity Bioanalyzer
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