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<p>Genomic loci positioning by sequencing (GPSeq) is a genome-wide method for mapping the radial organization of the genome in the nucleus of eukaryotic cells. GPSeq relies on in situ digestion of chromatin with a restriction enzyme that gradually diffuses inward from the nuclear periphery, followed by ligation of sequencing adapters to the digested restriction enzyme sites and library preparation for high-throughput sequencing. In parallel, ligation of labeled imaging adapters to the digested restriction enzyme recognition sites enables monitoring of the progression of radial digestion by fluorescence microscopy, providing an essential internal quality control before proceeding with sequencing. By comparing samples in which chromatin has been digested for increasing time intervals, a GPSeq score is calculated for every genomic bin into which the genome is arbitrarily divided, and genome-wide radial maps are generated with a resolution as high as 25 kb. These maps allow exploration of the radial distribution of (epi)genomic features, gene expression levels, mutational landscapes, and genomic profiles of DNA damage, when integrated with other omic data. Here, we present a detailed step-by-step protocol for performing GPSeq and preprocessing GPSeq data. The entire protocol requires ~2 weeks from the start of sample preparation to having ready-to-sequence libraries and intermediate levels of expertise in molecular biology, genomics and microscopy.</p>

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GPSeq maps the radial organization of eukaryotic genomes along the nuclear periphery–center axis

  • 期刊论文

  • 发表日期：

    2026年1月19日

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  • Wing Hin Yip,
  • Kaja Harton,
  • Ilaria Castiglioni,
  • Britta A. M. Bouwman,
  • Carlos Jiménez,
  • Emily Georgiades,
  • Luuk Harbers,
  • Wenjing Kang,
  • Erik Wernersson,
  • Nicola Crosetto,
  • Magda Bienko

摘要

Genomic loci positioning by sequencing (GPSeq) is a genome-wide method for mapping the radial organization of the genome in the nucleus of eukaryotic cells. GPSeq relies on in situ digestion of chromatin with a restriction enzyme that gradually diffuses inward from the nuclear periphery, followed by ligation of sequencing adapters to the digested restriction enzyme sites and library preparation for high-throughput sequencing. In parallel, ligation of labeled imaging adapters to the digested restriction enzyme recognition sites enables monitoring of the progression of radial digestion by fluorescence microscopy, providing an essential internal quality control before proceeding with sequencing. By comparing samples in which chromatin has been digested for increasing time intervals, a GPSeq score is calculated for every genomic bin into which the genome is arbitrarily divided, and genome-wide radial maps are generated with a resolution as high as 25 kb. These maps allow exploration of the radial distribution of (epi)genomic features, gene expression levels, mutational landscapes, and genomic profiles of DNA damage, when integrated with other omic data. Here, we present a detailed step-by-step protocol for performing GPSeq and preprocessing GPSeq data. The entire protocol requires ~2 weeks from the start of sample preparation to having ready-to-sequence libraries and intermediate levels of expertise in molecular biology, genomics and microscopy.