EXPERIMENT_TITLE: GSM5402791: Sample 2_cHi-C day5 sgCtrl rep2; Homo sapiens; Hi-C
EXPERIMENT_DESIGN_LIBRARY_DESCRIPTOR_LIBRARY_CONSTRUCTION_PROTOC: cHi-C libraries were prepared from CRISPRi sgCtrl or sgLNCSOX17 EN cells. 5 x 106 ZIP13K2-derived EN cells were harvested and washed with ice cold DPBS. Cell lysis, NlaIII (NEB, R0125) digestion and proximity-ligation was performed according to the Franke et al. protocol8 with minor changes. Adaptors were added to DNA fragments and amplified according to Agilent Technologies instructions for Illumina sequencing. The library was hybridized to the custom-designed SureSelect probes (Agilent Technologies, 5190-4806 / 3253271) (s. probe list in Suppl. Table 3) and indexed for sequencing of 200 M fragments /sample (100 bp paired-end) following the Agilent instructions. Capture Hi-C experiments were performed as biological duplicates.
STUDY_DESCRIPTOR_STUDY_ABSTRACT: Long noncoding RNAs (lncRNAs) have emerged as fundamental regulators in various biological processes, including embryonic development and cellular differentiation. Despite much progress over the past decade, the genome-wide annotation of lncRNAs remains incomplete and many known non-coding loci are poorly characterized. Here we report the discovery of a previously not annotated lncRNA that is transcribed 230 kb upstream of the SOX17 gene and located within the same topologically isolated domain. We therefore termed it LNCSOX17 and show that it is induced following SOX17 activation, but is more tightly restricted to early definitive endoderm than SOX17. Notably, loss of LNCSOX17 affects crucial functions independent of SOX17 and leads to an aberrant endodermal transcriptome, signaling pathway deregulation, and epithelial to mesenchymal transition defects. Consequently, cells lacking LNCSOX17 cannot further differentiate into more mature endodermal cell types. Our study identified LNCSOX17 and demonstrates its essential role as a new actor in early human endoderm, thereby further expanding the list of functionally important non-coding regulators. Overall design: cHi-C seq, 4C seq, RNAseq, ChIPseq and Oxford Nanopore cDNA seq were performed on hiPSCs/hESCs (day0), hiPSCs/hESCs derived endoderm (day5), intermediate differentiation stage (day3) or pancreatic progenitors (day9). Cells were grown as described in the methods section or in the protocol sections below. Libraries have been performed following manufacturer instructions and how described in the methods section or below.
SAMPLE_TITLE: Sample 2_cHi-C day5 sgCtrl rep2
SAMPLE_SAMPLE_ATTRIBUTES_SAMPLE_ATTRIBUTE: [{"TAG": "source_name", "VALUE": "Capture Hi-C seq of day5 endoderm derived CRISPRi cells expressing sgCtrl gRNA"}, {"TAG": "cell line/type", "VALUE": "ZIP13K2 - hiPSCs"}, {"TAG": "molecule subtype", "VALUE": "Enriched fragmented gDNA"}]
Pool_Member_@attributes_sample_title: Sample 2_cHi-C day5 sgCtrl rep2
RUN_SET_RUN_Pool_Member_@attributes_sample_title: Sample 2_cHi-C day5 sgCtrl rep2
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