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. 2021 Aug 10;13:128. doi: 10.1186/s13073-021-00941-8

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© The Author(s) 2021

Open AccessThis article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.

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Fig. 1 — Single nuclei isolation from frozen human islet samples. A Experimental workflow of isolation of nuclei from frozen cultured human islets (donor 1) and human islet dispersion into single cells (donor 2). B Isolated nuclei from frozen human islets (donor 1) and dissociated human islet cells from fresh intact islets (donor 2, whole cells) were imaged by DAPI/Phase microscopy. Left image: Intact nuclei stained in blue without cytoplasm surrounding the isolated nucleus . Right image: Nuclei stained in blue surrounded by cytoplasm of human islet cells. Scale bar is 25 μm. C Western blot analysis of human pancreatic islet cells (whole cell lysate; WCL, obtained from donor 2), supernatant collected after the first centrifuge step (cytosolic fraction A; Cyt A) and second centrifuge step (cytosolic fraction B; Cyt B) of the nuclear isolation protocol, and isolated nuclei (nuclear fraction; Nuc, obtained from Donor 1). LaminA/C is a nuclear marker; GAPDH is a cytoplasmic marker. D RNA was isolated from islet cell nuclei and analyzed by Bioanalyzer. Representative electropherogram showing two major peaks (18S and 28S rRNA) indicates minimal degradation of total RNA. The X-axis indicates the size of RNA fragments (nt: nucleotides) and the Y-axis represents the intensity of the fluorescence signal (FU, fluorescence unit). E Yield (μg, left Y-axis, dots) and RNA Integrity Number (RIN, right Y-axis, squares) of RNA isolated from six technical replicates across three independent experiments. Data are represented as mean ± SEM