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Figure 2

Schematic description of the SELEX-seq approach. The basic procedures of SELEX-seq include three steps. The first step is to design and synthesize an ssDNA library and then convert ssDNA into dsDNA using primer extension. The ssDNA library contained all possible oligonucleotides in random sequence of length k. The length of k can be determined according to the nucleotide numbers of DNA-binding consensus bound by a TF protein of interest. The second step is to bind TF proteins to randomized dsDNA library and isolate the protein-bound dsDNAs from binding reaction using gel mobility shift assay (also called electrophoresis mobility shift assay, EMSA) (Zykovich et al. 2009), affinity chromatography (Zykovich et al. 2009), or TF protein-coupled microwell plate (Jolma et al. 2010). The isolated dsDNAs are amplified by PCR to prepare a new library for the next round of the selection process. Through several rounds of repeated screening, the TF protein-bound dsDNAs were enriched. To find all sequences that can be bound by a TF protein with various binding affinities, especially sequences with low affinity, the isolated dsDNAs from each round of selection can be separately collected for sequencing (Zykovich et al. 2009). The third step of SELEX-seq is to sequence the bound dsDNAs with massively parallel DNA sequencing techniques, such as Illumina SOLEXA. The sequencing reads are filtered with filters including only A, C, G, and T letters allowed, valid bar code, and constant regions and unique random regions. If multiple DNA samples are simultaneously sequenced, the filtered reads are sorted according to bar code sequence. The qualified reads data are then used to perform subsequent bioinformatics analysis, including finding motifs or position weight matrix (PWM) models with some typical algorithms for this purpose, such as multiple EM for motif elicitation (MEME) (Bailey & Elkan 1994). A more detailed experimental and computational procedure to infer parameters of TF-DNA interaction from SELEX experiments was described by some studies (Djordjevic & Sengupta 2006). Full colour version of this figure available via http://dx.doi.org/10.1530/JME-11-0010.

This Article

  1. Published online before print March 9, 2011, doi: 10.1530/JOE-11-0010 J Endocrinol vol. 210 no. 1 15-27
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