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Comparative framework and adaptation of ACME HS approach to single cell isolation from fresh-frozen endocrine tissue.

ABSTRACT: Comparative framework and adaptation of ACME HS approach to single cell isolation from fresh-frozen endocrine tissue.

PROVIDER: PRJNA1100946 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR28712978_1.fastq.gz	Fastqsanger.gz
	SRR28712978_2.fastq.gz	Fastqsanger.gz
	SRR28712979_1.fastq.gz	Fastqsanger.gz
	SRR28712979_2.fastq.gz	Fastqsanger.gz
	SRR28712980_1.fastq.gz	Fastqsanger.gz

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Comparative framework and adaptation of ACME HS approach to single cell isolation from fresh-frozen endocrine tissue.

Project description:Current dissociation methods for scRNA-seq studies of solid tissues do not guarantee intact single-cell isolation from fresh frozen samples, especially for sensitive and heterogeneous endocrine tissues. Here, we adapted the acetic-methanol dissociation method – ACME High Soult (ACME HS) to isolate intact single cells from fresh-frozen endocrine tumor samples. We compared enzymatic, ACME HS, and nuclear isolation methods ability to preserve major cell types and gene expression. We demonstrated that ACME HS dissociates and fixes cells, preserving cell morphology and high RNA integrity. This renders ACME HS a valuable alternative in the scRNA-seq protocols for challenging tissues.

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Comparative framework and adaptation of ACME HS approach to single cell isolation from fresh-frozen endocrine tissue [dataset 1]

Project description:Current dissociation methods for scRNA-seq studies of solid tissues do not guarantee intact single-cell isolation from fresh frozen samples, especially for sensitive and heterogeneous endocrine tissues. Here, we adapted the acetic-methanol dissociation method – ACME High Salt (ACME HS) to isolate intact single cells from fresh-frozen endocrine tumor samples. We compared enzymatic, ACME HS, and nuclear isolation methods ability to preserve major cell types and gene expression. We demonstrated that ACME HS dissociates and fixes cells, preserving cell morphology and high RNA integrity. This renders ACME HS a valuable alternative in the scRNA-seq protocols for challenging tissues.

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Comparative framework and adaptation of ACME HS approach to single cell isolation from fresh-frozen endocrine tissue [dataset 2]

Project description:Current dissociation methods for scRNA-seq studies of solid tissues do not guarantee intact single-cell isolation from fresh frozen samples, especially for sensitive and heterogeneous endocrine tissues. Here, we adapted the acetic-methanol dissociation method – ACME High Salt (ACME HS) to isolate intact single cells from fresh-frozen endocrine tumor samples. We compared enzymatic, ACME HS, and nuclear isolation methods ability to preserve major cell types and gene expression. We demonstrated that ACME HS dissociates and fixes cells, preserving cell morphology and high RNA integrity. This renders ACME HS a valuable alternative in the scRNA-seq protocols for challenging tissues.

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Comparative framework and adaptation of ACME HS approach to single cell isolation from fresh-frozen endocrine tissue [dataset 2]

Project description:Comparative framework and adaptation of ACME HS approach to single cell isolation from fresh-frozen endocrine tissue [dataset 2]

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Comparative framework and adaptation of ACME HS approach to single cell isolation from fresh-frozen endocrine tissue [dataset 1]

Project description:Comparative framework and adaptation of ACME HS approach to single cell isolation from fresh-frozen endocrine tissue [dataset 1]

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Project description:Single-cell sequencing technologies are revolutionizing biology, but are limited by the need of dissociating fresh samples that can only be fixed at later stages. We present ACME (ACetic-MEthanol) dissociation, a species-versatile cell dissociation approach that fixes cells as they are being dissociated. ACME-dissociated cells have high RNA integrity, can be cryopreserved multiple times, can be sorted by Fluorescence-Activated Cell Sorting (FACS) and are permeable, enabling combinatorial approaches of single-cell transcriptomics. ACME is based on cheap reagents and it can be done in most labs and even sampling trips. As a proof of principle, we have performed SPLiT-seq with ACME cells to obtain around ~35K cells from two planarian species and identified all previously described cell types in similar proportions. This technique allows fixed, dissociated cells to be obtained from diverse organisms that can be cryopreserved and subjected to combinatorial barcoding methods for single-cell transcriptomics and thus will accelerate our knowledge of cell types across the tree of life.

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Project description:The epidemic character of community-associated methicillin resistant Staphylococcus aureus (CA-MRSA), especially the geographically widespread clone USA300, is poorly understood. USA300 isolates carry a type IV staphylococcal chromosomal cassette mec (SCCmec) element conferring -lactam antibiotic class resistance and a putative pathogenicity island, ACME (arginine catabolic mobile element). Physical linkage between SCCmec and ACME suggests that selection for antibiotic resistance and for pathogenicity may be interconnected. We constructed isogenic mutants containing deletions of SCCmec and ACME in a USA300 clinical isolate to determine the role of these elements in a rabbit model of bacteremia. We found that deletion of type IV SCCmec did not affect competitive fitness, whereas deletion of ACME significantly attenuated pathogenicity or fitness of USA300. These data are consistent with a model in which ACME enhances growth and survival of USA300, allowing for genetic "hitchhiking" of SCCmec. SCCmec in turn protects against exposure to Î² -lactams. Keywords: Wild type control vs mutant Wild type untreated in triplicate is compared to three mutants in triplicate in both exponential and stationary growth phases, totalling 24 samples

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