Psychrophilic proteases dramatically reduce single-cell RNA-seq artifacts: a molecular atlas of kidney development. Adam, M, Potter, A., & Potter, S. Development, 144(19):3625–3632, October, 2017. doi abstract bibtex Single-cell RNA-seq is a powerful technique. Nevertheless, there are important limitations, including the technical challenges of breaking down an organ or tissue into a single-cell suspension. Invariably, this has required enzymatic incubation at 37°C, which can be expected to result in artifactual changes in gene expression patterns. Here, we describe a dissociation method that uses a protease with high activity in the cold, purified from a psychrophilic microorganism. The entire procedure is carried out at 6°C or colder, at which temperature mammalian transcriptional machinery is largely inactive, thereby effectively ‘freezing in’ the in vivo gene expression patterns. To test this method, we carried out RNA-seq on 20,424 single cells from postnatal day 1 mouse kidneys, comparing the results of the psychrophilic protease method with procedures using 37°C incubation. We show that the cold protease method provides a great reduction in gene expression artifacts. In addition, the results produce a single-cell resolution gene expression atlas of the newborn mouse kidney, an interesting time in development when mature nephrons are present yet nephrogenesis remains extremely active.
@article{adam_psychrophilic_2017,
title = {Psychrophilic proteases dramatically reduce single-cell {RNA}-seq artifacts: a molecular atlas of kidney development},
volume = {144},
doi = {doi: 10.1242/dev.151142},
abstract = {Single-cell RNA-seq is a powerful technique. Nevertheless, there are important limitations, including the technical challenges of breaking down an organ or tissue into a single-cell suspension. Invariably, this has required enzymatic incubation at 37°C, which can be expected to result in artifactual changes in gene expression patterns. Here, we describe a dissociation method that uses a protease with high activity in the cold, purified from a psychrophilic microorganism. The entire procedure is carried out at 6°C or colder, at which temperature mammalian transcriptional machinery is largely inactive, thereby effectively ‘freezing in’ the in vivo gene expression patterns. To test this method, we carried out RNA-seq on 20,424 single cells from postnatal day 1 mouse kidneys, comparing the results of the psychrophilic protease method with procedures using 37°C incubation. We show that the cold protease method provides a great reduction in gene expression artifacts. In addition, the results produce a single-cell resolution gene expression atlas of the newborn mouse kidney, an interesting time in development when mature nephrons are present yet nephrogenesis remains extremely active.},
number = {19},
journal = {Development},
author = {Adam, M and Potter, AS and Potter, SS},
month = oct,
year = {2017},
pages = {3625--3632},
}
Downloads: 0
{"_id":"7gQYpNtHcLqw8dgWR","bibbaseid":"adam-potter-potter-psychrophilicproteasesdramaticallyreducesinglecellrnaseqartifactsamolecularatlasofkidneydevelopment-2017","downloads":0,"creationDate":"2018-02-27T22:34:36.972Z","title":"Psychrophilic proteases dramatically reduce single-cell RNA-seq artifacts: a molecular atlas of kidney development","author_short":["Adam, M","Potter, A.","Potter, S."],"year":2017,"bibtype":"article","biburl":"https://api.zotero.org/users/3649949/collections/E8UNN9WZ/items?key=bHPKkSJPRkAm4hCOP7l6ghDj&format=bibtex&limit=100","bibdata":{"bibtype":"article","type":"article","title":"Psychrophilic proteases dramatically reduce single-cell RNA-seq artifacts: a molecular atlas of kidney development","volume":"144","doi":"doi: 10.1242/dev.151142","abstract":"Single-cell RNA-seq is a powerful technique. Nevertheless, there are important limitations, including the technical challenges of breaking down an organ or tissue into a single-cell suspension. Invariably, this has required enzymatic incubation at 37°C, which can be expected to result in artifactual changes in gene expression patterns. Here, we describe a dissociation method that uses a protease with high activity in the cold, purified from a psychrophilic microorganism. The entire procedure is carried out at 6°C or colder, at which temperature mammalian transcriptional machinery is largely inactive, thereby effectively ‘freezing in’ the in vivo gene expression patterns. To test this method, we carried out RNA-seq on 20,424 single cells from postnatal day 1 mouse kidneys, comparing the results of the psychrophilic protease method with procedures using 37°C incubation. We show that the cold protease method provides a great reduction in gene expression artifacts. In addition, the results produce a single-cell resolution gene expression atlas of the newborn mouse kidney, an interesting time in development when mature nephrons are present yet nephrogenesis remains extremely active.","number":"19","journal":"Development","author":[{"propositions":[],"lastnames":["Adam"],"firstnames":["M"],"suffixes":[]},{"propositions":[],"lastnames":["Potter"],"firstnames":["AS"],"suffixes":[]},{"propositions":[],"lastnames":["Potter"],"firstnames":["SS"],"suffixes":[]}],"month":"October","year":"2017","pages":"3625–3632","bibtex":"@article{adam_psychrophilic_2017,\n\ttitle = {Psychrophilic proteases dramatically reduce single-cell {RNA}-seq artifacts: a molecular atlas of kidney development},\n\tvolume = {144},\n\tdoi = {doi: 10.1242/dev.151142},\n\tabstract = {Single-cell RNA-seq is a powerful technique. Nevertheless, there are important limitations, including the technical challenges of breaking down an organ or tissue into a single-cell suspension. Invariably, this has required enzymatic incubation at 37°C, which can be expected to result in artifactual changes in gene expression patterns. Here, we describe a dissociation method that uses a protease with high activity in the cold, purified from a psychrophilic microorganism. The entire procedure is carried out at 6°C or colder, at which temperature mammalian transcriptional machinery is largely inactive, thereby effectively ‘freezing in’ the in vivo gene expression patterns. To test this method, we carried out RNA-seq on 20,424 single cells from postnatal day 1 mouse kidneys, comparing the results of the psychrophilic protease method with procedures using 37°C incubation. We show that the cold protease method provides a great reduction in gene expression artifacts. In addition, the results produce a single-cell resolution gene expression atlas of the newborn mouse kidney, an interesting time in development when mature nephrons are present yet nephrogenesis remains extremely active.},\n\tnumber = {19},\n\tjournal = {Development},\n\tauthor = {Adam, M and Potter, AS and Potter, SS},\n\tmonth = oct,\n\tyear = {2017},\n\tpages = {3625--3632},\n}\n\n","author_short":["Adam, M","Potter, A.","Potter, S."],"key":"adam_psychrophilic_2017","id":"adam_psychrophilic_2017","bibbaseid":"adam-potter-potter-psychrophilicproteasesdramaticallyreducesinglecellrnaseqartifactsamolecularatlasofkidneydevelopment-2017","role":"author","urls":{},"metadata":{"authorlinks":{}},"downloads":0},"search_terms":["psychrophilic","proteases","dramatically","reduce","single","cell","rna","seq","artifacts","molecular","atlas","kidney","development","adam","potter","potter"],"keywords":[],"authorIDs":[],"dataSources":["n5rzo9bDqWGobfs4D"]}