Comparison of optimized methodologies for isolating nuclei from esophageal tissue
Comparison of optimized methodologies for isolating nuclei from esophageal tissue
Single-nuclei RNA sequencing (snRNA-seq) allows single cell-based analysis in frozen tissue, ameliorating cell recovery biases associated with enzymatic dissociation methods. We present two optimised methods for isolating and sequencing nuclei from oesophageal tissue using a commercial EZ and citric acid-based (CA) method. Despite high endogenous RNase activity, these protocols produced libraries of expected fragment length (average length EZ = 745 bp, CA = 1232 bp) with comparable complexity (median Transcript/Gene number, EZ = 496/254, CA = 483/256). CA nuclei showed a higher proportion of ribosomal gene reads, potentially reflecting co-isolation of nuclei and adherent ribosomes. We identified 11 cell lineages in the combined datasets, with differences in cell type recovery between the two methods, providing utility dependent on experimental needs.
RNA, esophagus, nuclei, sequencing, single cell, tissue sampling, transcriptome
104-109
Kimbley, Lucy
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Parker, Rachel
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Harrington, Jack
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Walker, Robert, Charles
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Grace, Ben
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West, Jonathan
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Underwood, Timothy
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Rose-Zerilli, Matthew
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14 February 2022
Kimbley, Lucy
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Parker, Rachel
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Harrington, Jack
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Walker, Robert, Charles
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Grace, Ben
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West, Jonathan
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Underwood, Timothy
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Rose-Zerilli, Matthew
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Kimbley, Lucy, Parker, Rachel, Harrington, Jack, Walker, Robert, Charles, Grace, Ben, West, Jonathan, Underwood, Timothy and Rose-Zerilli, Matthew
(2022)
Comparison of optimized methodologies for isolating nuclei from esophageal tissue.
Biotechniques, 72 (3), .
(doi:10.2144/btn-2021-0036).
Abstract
Single-nuclei RNA sequencing (snRNA-seq) allows single cell-based analysis in frozen tissue, ameliorating cell recovery biases associated with enzymatic dissociation methods. We present two optimised methods for isolating and sequencing nuclei from oesophageal tissue using a commercial EZ and citric acid-based (CA) method. Despite high endogenous RNase activity, these protocols produced libraries of expected fragment length (average length EZ = 745 bp, CA = 1232 bp) with comparable complexity (median Transcript/Gene number, EZ = 496/254, CA = 483/256). CA nuclei showed a higher proportion of ribosomal gene reads, potentially reflecting co-isolation of nuclei and adherent ribosomes. We identified 11 cell lineages in the combined datasets, with differences in cell type recovery between the two methods, providing utility dependent on experimental needs.
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btn-2021-0036
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Accepted/In Press date: 17 January 2022
e-pub ahead of print date: 14 February 2022
Published date: 14 February 2022
Keywords:
RNA, esophagus, nuclei, sequencing, single cell, tissue sampling, transcriptome
Identifiers
Local EPrints ID: 454628
URI: http://eprints.soton.ac.uk/id/eprint/454628
ISSN: 0736-6205
PURE UUID: 4c47638f-ec88-4394-b94a-3e166ccdc2e2
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Date deposited: 17 Feb 2022 17:39
Last modified: 17 Mar 2024 03:29
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Author:
Lucy Kimbley
Author:
Rachel Parker
Author:
Jack Harrington
Author:
Robert, Charles Walker
Author:
Ben Grace
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