Transposase-assisted tagmentation of RNA/DNA hybrid duplexes

  1. Bo Lu
  2. Liting Dong
  3. Danyang Yi
  4. Meiling Zhang
  5. Chenxu Zhu
  6. Xiaoyu Li
  7. Chengqi Yi  Is a corresponding author
  1. State Key Laboratory of Protein and Plant Gene Research, School of Life Sciences, Peking University, China
  2. Peking-Tsinghua Center for Life Sciences, Peking University, China
  3. Department of Chemical Biology and Synthetic and Functional Biomolecules Center, College of Chemistry and Molecular Engineering, Peking University, China
3 figures, 1 table and 6 additional files

Figures

Figure 1 with 1 supplement
Tn5 transposome has direct tagmentation activity on RNA/DNA hybrid duplexes.

(a) Crystal structure of a single subunit of E. coli Tn5 Transposase (PDB code 1MM8) complexed with ME DNA duplex, and zoom-in views of the conserved catalytic core of Tn5 transposase, HIV-1 …

Figure 1—source data 1

qPCR Ct values of tagmentation products of samples under different conditions in Figure 1d and e.

https://cdn.elifesciences.org/articles/54919/elife-54919-fig1-data1-v2.xlsx
Figure 1—figure supplement 1
Tagmentation activity of Tn5 transposome on RNA/DNA hybrids.

(a) Denaturing (8 M urea) polyacrylamide gel analysis of reverse transcription products of an in vitro transcribed mRNA (IRF9). Lane 1: ssRNA marker. Lane 2: in vitro transcribed mRNA (IRF9). Lane 3 …

Figure 2 with 1 supplement
Workflow and evaluation of TRACE-seq.

(a) Workflow of TRACE-seq. (b) Gene expression, measured by three technical replicates of TRACE-seq with 200 ng total RNA as input, are shown as scatter plots in the upper right half. Pearson's …

Figure 2—source data 1

Distribution of reads across known genome features for NEBNext Ultra II RNA kit, Smart-seq2 and TRACE-seq with different amount of RNA as input.

https://cdn.elifesciences.org/articles/54919/elife-54919-fig2-data1-v2.xls
Figure 2—figure supplement 1
Quality assessment of TRACE-seq.

(a) Gene expression measured by two technical replicates of TRACE-seq with 20 ng total RNA as input are shown as scatter plots. Pearson's product-moment correlations are displayed in the upper left …

Performance of TRACE-seq in differential expression analysis.

(a) Volcano plot showing differential expressed genes between undifferentiated and differentiated mESCs detected by NEBNext Ultra II RNA kit and TRACE-seq. Significantly up-regulated and …

Tables

Appendix 1—key resources table
Reagent type
(species) or resource
DesignationSource or
reference
IdentifiersAdditional
information
Cell line (Homo-sapiens)HEK293TAmerican Type Culture CollectionCat#: CRL-11268, RRID:CVCL_1926
AntibodyMouse anti-DNA-RNA Hybrid [S9.6] AntibodyKerafastCat#: ENH001, RRID:AB_26874631:2000
AntibodyAntibody Anti-mouse-IgG-HRPCWBiotechCat#: CW0102, RRID:AB_27369971:3000
Recombinant DNA reagentCLuc Control Template NEBCat#: E2060S
Sequence-based reagentCLuc Control_FThis paperPCR primersTAATACGACTCACTATAGGG
Sequence-based reagentCLuc Control_RThis paperPCR primersTTAGCTTCACAGGAAGTTGG
Sequence-based reagentGAPDH-qFWDThis paperPCR primersGCATCCTGGGCTACACTGAG
Sequence-based reagentGAPDH-qRVSThis paperPCR primersAAAGTGGTCGTTGAGGGCAA
Sequence-based reagentACTB-qFWDThis paperPCR primersAGTCATTCCAAATATGAGATGCGTT
Sequence-based reagentACTB-qRVSThis paperPCR primersTGCTATCACCTCCCCTGTGT
Sequence-based reagentCYC1-qFWDThis paperPCR primersCACCATAAAGCGGCACAAGT
Sequence-based reagentCYC1-qRVSThis paperPCR primersCAGGATGGCAAGCAGACACT
Sequence-based reagentTn5ME-Adoi: 10.1186/gb-2010-11-12-r119Transposon adaptor oligonucleotidesTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
Sequence-based reagentTn5ME-Bdoi: 10.1186/gb-2010-11-12-r119Transposon adaptor oligonucleotidesGTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG
Sequence-based reagentTn5MErevdoi: 10.1186/gb-2010-11-12-r119Transposon adaptor oligonucleotidesCTGTCTCTTATACACATCT
Sequence-based reagentTn5_qFWDThis paperPCR primersAATGATACGGCGACCACCGAGATCTACACTCGTCGGCAGCGTC
Sequence-based reagentTn5_qRVSThis paperPCR primersCAAGCAGAAGACGGCATACGAGATGTCTCGTGGGCTCGG
Sequence-based reagentTSOdoi:10.1038/nprot.2014.006Template switch primerAAGCAGTGGTATCAACGCAGAGTACATrGrG+G
Sequence-based reagentISPCR oligodoi:10.1038/nprot.2014.006PCR primersAAGCAGTGGTATCAACGCAGAGT
Sequence-based reagentoligo dT(23)VN primerNEBCat#: S1327S
Sequence-based reagentRandom primer mixNEBCat#: S1330S
Sequence-based reagentN501 primerIlluminaPCR primers for sequencing
Sequence-based reagentN701-N712 primersIlluminaPCR primers for sequencing
Commercial assay or kit TRIzolInvitrogen Cat#: 15596018
Commercial assay or kitBlood & Cell Culture DNA Midi KitQiagen Cat#: 13343
Commercial assay or kitMAXIscript T7 Transcription KitInvitrogenCat#: AM1314M
Commercial assay or kitSUPERase-In RNase InhibitorInvitrogen Cat#: AM2696
Commercial assay or kitQuant-iT PicoGreen dsDNA Assay KitInvitrogenCat#: P11496
Commercial assay or kitAceQ Universal SYBR qPCR Master MixVazymeCat#: Q511-02
Commercial assay or kitpEASY-Blunt Zero Cloning KitTransGenCat#: CB501-01
Commercial assay or kitNEBNext Q5 Hot Start HiFi PCR Master MixNEBCat#: M0544
Commercial assay or kitAgencourt AMPure XP beadsBeckman CoulterCat#: A63882
Commercial assay or kitRNAClean XP beadsBeckman CoulterCat#: A63987
Commercial assay or kitQubit dsDNA HS Assay kitInvitrogenCat#: Q33230
Commercial assay or kitDNF-474 High Sensitivity NGS Fragment Analysis KitAgilentCat#: DNF-473-1000
Commercial assay or kitNEBNext Ultra II RNA Library Prep Kit for IlluminaNEBCat#: E7770S
Commercial assay or kitDynabeads Oligo(dT)25InvitrogenCat#: 61005
Commercial assay or kitKAPA HiFi HotStart ReadyMixKAPA BiosystemsCat#: KK2601
Commercial assay or kitTransDetect PCR Mycoplasma Detection KitTransGenCat#: FM311-01
Commercial assay or kitRNA 6000 Pico kits (Agilent TechnologiesAgilentCat#: 5067-1513
Peptide, recombinant protein DNase INEBCat#: M0303S
Peptide, recombinant proteinSuperScript IV reverse transcriptaseInvitrogen Cat#: 12594100
Peptide, recombinant proteinSuperScript II reverse transcriptaseInvitrogenCat#: 18064022
Peptide, recombinant proteinRNase HNEBCat#: M0297
Peptide, recombinant proteinTruePrep Tagment EnzymeVazymeCat#: S601-01
Peptide, recombinant proteinBst 3.0 DNA PolymeraseNEBCat#: M0374S
Chemical compound, drugPEG200SigmaCat#: 88440
Chemical compound, drugPEG8000SigmaCat#: 89510
Software, algorithmTrim Galorehttp://www.bioinformatics.babraham.ac.uk/projects/trim_galore/RRID:SCR_011847v0.6.4_dev
Software, algorithmSTARPMID:23104886RRID:SCR_015899v2.7.1a
Software, algorithmbowtie2https://doi.org/10.1038/nmeth.1923RRID:SCR_005476v2.2.9
Software, algorithmSamtoolshttp://samtools.sourceforge.net/RRID:SCR_002105v1.9
Software, algorithmcuffnormPMID:20436464RRID:SCR_014597v2.2.1
Software, algorithmQoRTshttps://doi.org/10.1186/s12859-015-0670-5RRID:SCR_018665v1.1.6
Software, algorithmRseQCPMID:22743226RRID:SCR_005275v2.6.4
Software, algorithmPicard Toolshttp://broadinstitute.github.io/picard/RRID:SCR_006525v2.20.6
Software, algorithmPreseqPMID:23435259RRID:SCR_018664v2.0.0
Software, algorithmRStudiohttps://rstudio.com/RRID:SCR_0004321.2.5033
Software, algorithmIntegrative Genomics Viewerhttp://software.broadinstitute.org/software/igv/RRID:SCR_011793v2.4.16

Additional files

Supplementary file 1

Quality control of the sequencing results using different enzymes for strand extension and PCR in TRACE-seq library construction procedure.

https://cdn.elifesciences.org/articles/54919/elife-54919-supp1-v2.xlsx
Supplementary file 2

Quality control of the sequencing results using NEBNext kit and TRACE-seq.

https://cdn.elifesciences.org/articles/54919/elife-54919-supp2-v2.xlsx
Supplementary file 3

Quality control of the sequencing results using Smart-seq2 and TRACE-seq.

https://cdn.elifesciences.org/articles/54919/elife-54919-supp3-v2.xlsx
Supplementary file 4

Costs of RNA-seq constructed by NEBNext Ultra II RNA kit, TRACE-seq and Smart-seq2.

https://cdn.elifesciences.org/articles/54919/elife-54919-supp4-v2.xlsx
Supplementary file 5

List of housekeeping genes.

https://cdn.elifesciences.org/articles/54919/elife-54919-supp5-v2.xlsx
Transparent reporting form
https://cdn.elifesciences.org/articles/54919/elife-54919-transrepform-v2.pdf

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