Daniel:Protocols/CPTSeq: Difference between revisions
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>Djacobse (Created page with "=Contiguity Preserving Transposition Sequencing (CPT-Seq)= Back to Main '''Notes''' *Original Protocol from [http://www.nature.com/ng/journal/v46/n12/ful...") |
>Djacobse |
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'''Notes''' | '''Notes''' | ||
*Original Protocol from [http://www.nature.com/ng/journal/v46/n12/full/ng.3119.html Amini et al] | *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]] | *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 |
Latest revision as of 20:29, 1 February 2016
Contiguity Preserving Transposition Sequencing (CPT-Seq)[edit]
Notes
- Original Protocol from Amini et al
- Input to this protocol should be high molecular weight DNA (as little fragmentation as possible); DNA Extraction Protocol
Transposon Complex Assembly[edit]
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[edit]
- 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[edit]
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