Human Thymus single cell dissociation protocol - Teichmann Lab v1

2021 ◽  
Author(s):  
Jongeun Park ◽  
Veronika Kedlian ◽  
Chenqu Suo ◽  
Liam Bolt ◽  
Alexander Steemers ◽  
...  
Keyword(s):  
Single Cell ◽  
Tissue Preservation ◽  
Cell Dissociation ◽  
Sanger Institute ◽  
Enzymatic Dissociation ◽  
Human Thymus

This Protocol is intended for human Thymus single cell dissociation. It includes tissue preservation and handling, Enzymatic dissociation, FACS an MACS enrichments. Developed In the Teichmann lab at the Sanger institute, Wellcome Gemone Campus, UK VK, CS and JP contributed equally

Single-Cell Transcriptomics Reveals Discrete Steps in Regulatory T Cell Development in the Human Thymus

2021 ◽  
pp. ji2100506
Author(s):  
Florencia Morgana ◽  
Rianne Opstelten ◽  
Manon C. Slot ◽  
Andrew M. Scott ◽  
René A. W. van Lier ◽  
...  
Keyword(s):  
T Cell ◽  
Single Cell ◽  
Regulatory T Cell ◽  
T Cell Development ◽  
Cell Development ◽  
Human Thymus

scholarly journals Novel fluid shear-based dissociation device for improved single cell dissociation of spheroids and cell aggregates

2017 ◽  
Vol 34 (1) ◽  
pp. 293-298 ◽  
Author(s):  
Ursula L. Triantafillu ◽  
Jaron N. Nix ◽  
Yonghyun Kim
Keyword(s):  
Single Cell ◽  
Cell Aggregates ◽  
Fluid Shear ◽  
Cell Dissociation

scholarly journals Single Cell Sequencing Reveals Glial Specific Responses to Tissue Processing & Enzymatic Dissociation in Mice and Humans

2020 ◽  
Author(s):  
Samuel E. Marsh ◽  
Tushar Kamath ◽  
Alec J. Walker ◽  
Lasse Dissing-Olesen ◽  
Timothy R. Hammond ◽  
...  
Keyword(s):  
Gene Expression ◽  
Single Cell ◽  
Human Tissue ◽  
Ex Vivo ◽  
Gene Expression Signature ◽  
Enzymatic Digestion ◽  
Careful Consideration ◽  
Post Mortem ◽  
Fresh Tissue ◽  
Enzymatic Dissociation

AbstractA key aspect of nearly all single cell experiments is the necessity to dissociate intact tissues into single cell suspensions for processing. While many protocols have been optimized for optimal cell yield, they have often overlooked the effects that dissociation can have on ex vivo gene expression changes during this process. Microglia, the brain’s resident macrophages, are a highly dynamic population that are extremely sensitive to their microenvironment and have been shown to dramatically alter their transcriptome upon stimulation. We demonstrate that use of enzymatic dissociation methods on mouse central nervous system (CNS) tissue induces an aberrant gene expression signature in microglia that can significantly confound downstream analysis. To minimize this issue, we developed a flexible protocol, that can be used with existing enzymatic protocols for fresh tissue, to eliminate artifactual gene expression while allowing for increased cell type diversity and yield. We demonstrate efficacy of this protocol in analysis of diverse CNS cell types and sorted myeloid populations while using enzymatic dissociation. Generation of new and reanalysis of previously published human brain single nucleus RNAseq (snRNA-seq) datasets reveal that a similar signature is also present in post-mortem tissue. Through novel snRNA-seq analysis of acutely-resected neurosurgical tissue we demonstrate that this signature can be induced in human tissue due to technical differences in sample processing. These results provide key insight into the potential confounds of enzymatic digestion and provide a solution to allow for enzymatic digestion for scRNA-seq while avoiding ex vivo transcriptional artifacts. Analysis of human tissue reveals potential for artifacts in current and future snRNA-seq datasets that will require deeper analysis and careful consideration to separate true biology from artifacts related to post-mortem processes.

scholarly journals Human skin single cell dissociation v1 (protocols.io.ripd4dn)

protocols.io ◽  
2018 ◽  
Author(s):  
James Fletcher ◽  
Rachel Botting ◽  
Emily Stephenson ◽  
Peter Vegh ◽  
Muzlifah Haniffa
Keyword(s):  
Single Cell ◽  
Human Skin ◽  
Cell Dissociation

scholarly journals ACME dissociation: a versatile cell fixation-dissociation method for single-cell transcriptomics

2020 ◽  
Author(s):  
Helena García-Castro ◽  
Nathan J Kenny ◽  
Patricia Álvarez-Campos ◽  
Vincent Mason ◽  
Anna Schönauer ◽  
...  
Keyword(s):  
Single Cell ◽  
Cell Sorting ◽  
Cell Types ◽  
Cell Dissociation ◽  
Rna Integrity ◽  
Single Cell Sequencing ◽  
Sequencing Technologies ◽  
A Cell ◽  
Dissociated Cells ◽  
Cell Fixation

AbstractSingle-cell sequencing technologies are revolutionizing biology, but are limited by the need to dissociate fresh samples that can only be fixed at later stages. We present ACME (ACetic-MEthanol) dissociation, a 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 single-cell transcriptomic approaches. As a proof of principle, we have performed SPLiT-seq with ACME cells to obtain around ∼34K single cell transcriptomes from two planarian species and identified all previously described cell types in similar proportions. ACME is based on affordable reagents, can be done in most laboratories and even in the field, and thus will accelerate our knowledge of cell types across the tree of life.

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