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High-throughput sequencing SELEX for the determination of DNA-binding protein specificities in vitro

Pantier, Raphaël, Chhatbar, Kashyap, Alston, Grace, Lee, Heng Yang, Bird, Adrian·2022·STAR Protocols
DOI 10.1016/j.xpro.2022.101490PMC PMC9243297
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Graphical abstract CC BY · PubMed Central
Figure 1 Strategy for generating a double-stranded DNA library using a single-stranded DNA template and two flanking primers The initial SELEX library (cycle 0) contains a “random insert”, corresponding to all putative sequence motifs for DNA-binding proteins.
Figure 1 Strategy for generating a double-stranded DNA library using a single-stranded DNA template and two flanking primers The initial SELEX library (cycle 0) contains a “random insert”, corresponding to all putative sequence motifs for DNA-binding proteins.CC BY · PubMed Central
Figure 2 10% polyacrylamide gel showing the generation of three independent libraries of random oligonucleotides (cycle 0) to initiate the HT-SELEX protocol
Figure 2 10% polyacrylamide gel showing the generation of three independent libraries of random oligonucleotides (cycle 0) to initiate the HT-SELEX protocolCC BY · PubMed Central
Figure 3 10% polyacrylamide gel showing the optimization of PCR conditions to amplify enriched DNA from Ni Sepharose beads following a cycle of SELEX For this sample, we purified the product following to 12× PCR cycles, as it showed a robust amplification of the library (83 bp) without detectable heteroduplexes (see troubleshooting 1).
Figure 3 10% polyacrylamide gel showing the optimization of PCR conditions to amplify enriched DNA from Ni Sepharose beads following a cycle of SELEX For this sample, we purified the product following to 12× PCR cycles, as it showed a robust amplification of the library (83 bp) without detectable heteroduplexes (see troubleshooting 1).CC BY · PubMed Central
Figure 4 Strategy for generating HT-SELEX libraries for high-throughput sequencing Following PCR, each SELEX library contains Illumina sequencing adapters (P5/P7) and unique barcodes (i7 indexes).
Figure 4 Strategy for generating HT-SELEX libraries for high-throughput sequencing Following PCR, each SELEX library contains Illumina sequencing adapters (P5/P7) and unique barcodes (i7 indexes).CC BY · PubMed Central
Figure 5 10% polyacrylamide gel showing the generation of 9× HT-SELEX libraries (144 bp) for high-throughput sequencing
Figure 5 10% polyacrylamide gel showing the generation of 9× HT-SELEX libraries (144 bp) for high-throughput sequencingCC BY · PubMed Central
Figure 6 Bioanalyzer profile showing the successful elimination of PCR primers from HT-SELEX library pool following a 3× clean-up with KAPA Pure beads
Figure 6 Bioanalyzer profile showing the successful elimination of PCR primers from HT-SELEX library pool following a 3× clean-up with KAPA Pure beadsCC BY · PubMed Central
Figure 7 Enrichment of all possible 5-mer DNA motifs during the HT-SELEX protocol for SALL4 ZFC4 (blue) compared to negative control (gray) DNA motifs (k-mers) were divided into six categories of increasing A/T content. Error bars indicate the technical variability with independent SELEX libraries.
Figure 7 Enrichment of all possible 5-mer DNA motifs during the HT-SELEX protocol for SALL4 ZFC4 (blue) compared to negative control (gray) DNA motifs (k-mers) were divided into six categories of increasing A/T content. Error bars indicate the technical variability with independent SELEX libraries.CC BY · PubMed Central
Figure 8 10% polyacrylamide gel showing the optimization of the amount of DNA template to generate cycle 0 SELEX libraries 0.5 pmol of “Random library” per 50 μL PCR reaction (10 nM concentration) results in robust amplification of the library after 5× PCR cycles without detectable heteroduplexes.
Figure 8 10% polyacrylamide gel showing the optimization of the amount of DNA template to generate cycle 0 SELEX libraries 0.5 pmol of “Random library” per 50 μL PCR reaction (10 nM concentration) results in robust amplification of the library after 5× PCR cycles without detectable heteroduplexes.CC BY · PubMed Central
Figure 9 Rank comparison (Spearman R2) between k-mer abundance in sub-sampled libraries compared with original samples (≈3,000,000 reads per sample)
Figure 9 Rank comparison (Spearman R2) between k-mer abundance in sub-sampled libraries compared with original samples (≈3,000,000 reads per sample)CC BY · PubMed Central

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Research works printed but not linked: 12

  1. Ellington, 1990, In vitro selection of RNA molecules that bind specific ligands, Nature, 346, 818, 10.1038/346818a0 · DOI
  2. Gräslund, 2008, Protein production and purification, Nat. Methods, 5, 135, 10.1038/nmeth.f.202 · DOI
  3. Jolma, 2010, Multiplexed massively parallel SELEX for characterization of human transcription factor binding specificities, Genome Res., 20, 861, 10.1101/gr.100552.109 · DOI · Paper page
  4. Jolma, 2013, DNA-binding specificities of human transcription factors, Cell, 152, 327, 10.1016/j.cell.2012.12.009 · DOI
  5. Jolma, 2020, Binding specificities of human RNA-binding proteins toward structured and linear RNA sequences, Genome Res., 30, 962, 10.1101/gr.258848.119 · DOI
  6. Kanagawa, 2003, Bias and artifacts in multitemplate polymerase chain reactions (PCR), J. Biosci. Bioeng., 96, 317, 10.1016/S1389-1723(03)90130-7 · DOI
  7. Kielkopf, 2021, Expressing cloned genes for protein production, purification, and analysis, Cold Spring Harb. Protoc., 2021, 10.1101/pdb.top102129 · DOI
  8. Nitta, 2015, Conservation of transcription factor binding specificities across 600 million years of bilateria evolution, Elife, 4, e04837, 10.7554/eLife.04837 · DOI
  9. Roulet, 2002, High-throughput SELEX–SAGE method for quantitative modeling of transcription-factor binding sites, Nat. Biotechnol., 20, 831, 10.1038/nbt718 · DOI
  10. Slattery, 2011, Cofactor binding evokes latent differences in DNA binding specificity between Hox proteins, Cell, 147, 1270, 10.1016/j.cell.2011.10.053 · DOI
  11. Thompson, 2002, Heteroduplexes in mixed-template amplifications: formation, consequence and elimination by ‘reconditioning PCR, Nucleic Acids Res., 30, 2083, 10.1093/nar/30.9.2083 · DOI
  12. Tuerk, 1990, Systematic evolution of ligands by exponential enrichment: RNA ligands to bacteriophage T4 DNA polymerase, Science, 249, 505, 10.1126/science.2200121 · DOI

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Metadata

Authors
Pantier, R., Chhatbar, K., Alston, G., Lee, H. Y., & Bird, A.
Venue
STAR Protocols
Publisher
Elsevier BV
Published
2022-09
Volume
3
Issue
3
Pages
101490
ISSN
2666-1667
DOI
10.1016/j.xpro.2022.101490
PMC
PMC9243297
Cited by
28
References
2
Language
en
Licence
Funders
  • ERC (EC 694295 Gen-Epix)
  • European Research Council
  • University of Edinburgh
  • Simons Initiative for the Developing Brain
  • Wellcome Trust (092076, 107930, 203149)

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