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]

Back to Main

Notes

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.

  1. Mix Tn5 mosaic ends and complementary oligonucleotides in annealing buffer
    1. Annealing buffer: 10mM Tris-HCl, 1mM EDTA, 25 mM NaCl, pH 8.0
  2. Anneal using a themocycler with following parameters:
    1. 95C for 5 min
    2. decrease to 25C at -0.1C/s
  3. Transposons were then individually mixed at a 1:1 molar ratio with EZ-Tn5 at final stock concentration of 12.5 uM
  4. Incubate at 37C for 1 hour
  5. Assess quality using an 8% TBE gel
  6. Generate 96 transposome complexes
    1. 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
    2. Store at -20C
  7. Working stock is 2.5 uM and can be stored at -20C

Transposition of High MW gDNA[edit]

  1. Set up 96 transposition reactions on ice in low-DNA-binding PCR plate
    1. 1 ng Hi-MW gDNA
    2. 10 uL of 2X Nextera Tagment DNA buffer
    3. 8 uL water
  2. Add 2.5 pmol of each transposome complex to its respective well; mix gently by pipetting
  3. Incubate for 10 min at 55C in a thermocycler with a heated lid
  4. Stop transposition by adding 20 uL of 40 mM EDTA (pH 8.0) to each reaction
  5. Incubate for 15 min at 37C
  6. Pool 20 uL from each well into a plastic basin and gently rock for 5 min at 2 r.p.m. to mix well
  7. Dilute 1:25 (25 pg/uL -> 1 pg/uL) in 1X TE Buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0)
  8. 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
  9. To dissociate Tn5, add 2 uL of 1% SDS and gently mix by pipetting
  10. 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

  1. Add 10 uL of 5 uM i5 primer to its respective well after SDS treatment
  2. 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
  3. For PCR
    1. 72C for 3 min
    2. 98C for 30 sec
    3. 20 cycles
      1. 98C for 30 sec
      2. 63C for 30 sec
      3. 72C for 3 min
  4. Pool all 96 PCRs and purify
    1. 50 uL aliquots pooled
    2. Assess quality with High-Sensitivity Bioanalyzer