In situ polyadenylation enables spatial mapping of the total transcriptome

McKellar, David W.; Mantri, Madhav; Hinchman, Meleana M.; Parker, John S. L.; Sethupathy, Praveen; Cosgrove, Benjamin D.; Vlaminck, Iwijn De

doi:10.1101/2022.04.20.488964

Cited by 4 publications

(2 citation statements)

References 50 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…In particular, newly formed multinucleated muscle fibers are highly transcriptionally active and may well contribute significantly to the pro-regenerative transitional ECM that instructs MuSC function following skeletal muscle injury. Emerging technologies such as spatial RNA-sequencing, multiplexed proteomics, and single-cell protein analysis, will ideally further advance our understanding of the complex niche regulation of MuSCs ( Pham et al, 2021 ; McKellar et al, 2022 ; Wang et al, 2022 ). Importantly, compared to other relatively static stem cell niches, the microenvironment of MuSCs is highly dynamic and depends on the respective stage of muscle regeneration.…”

Section: Discussionmentioning

confidence: 99%

Extracellular matrix: Brick and mortar in the skeletal muscle stem cell niche

Schüler

Dumontier

et al. 2022

Front. Cell Dev. Biol.

View full text Add to dashboard Cite

The extracellular matrix (ECM) is an interconnected macromolecular scaffold occupying the space between cells. Amongst other functions, the ECM provides structural support to tissues and serves as a microenvironmental niche that conveys regulatory signals to cells. Cell-matrix adhesions, which link the ECM to the cytoskeleton, are dynamic multi-protein complexes containing surface receptors and intracellular effectors that control various downstream pathways. In skeletal muscle, the most abundant tissue of the body, each individual muscle fiber and its associated muscle stem cells (MuSCs) are surrounded by a layer of ECM referred to as the basal lamina. The core scaffold of the basal lamina consists of self-assembling polymeric laminins and a network of collagens that tether proteoglycans, which provide lateral crosslinking, establish collateral associations with cell surface receptors, and serve as a sink and reservoir for growth factors. Skeletal muscle also contains the fibrillar collagenous interstitial ECM that plays an important role in determining tissue elasticity, connects the basal laminae to each other, and contains matrix secreting mesenchymal fibroblast-like cell types and blood vessels. During skeletal muscle regeneration fibroblast-like cell populations expand and contribute to the transitional fibronectin-rich regenerative matrix that instructs angiogenesis and MuSC function. Here, we provide a comprehensive overview of the role of the skeletal muscle ECM in health and disease and outline its role in orchestrating tissue regeneration and MuSC function.

show abstract

Section: Discussionmentioning

confidence: 99%

Extracellular matrix: Brick and mortar in the skeletal muscle stem cell niche

Schüler

Dumontier

et al. 2022

Front. Cell Dev. Biol.

View full text Add to dashboard Cite

show abstract

“…Gene expression quantification of the Visium dataset was done using kallisto (0.48.0) and bustools (0.41.0) as previously described 62 . Cells were filtered with DropletUtils::emptyDrops and DropletUtils::defaultDrops as described.…”

Section: Spatial Transcriptomics Data Analysismentioning

confidence: 99%

Multi-species atlas resolves an axolotl limb development and regeneration paradox

Zhong

Aires

Tsissios

et al. 2023

Preprint

View full text Add to dashboard Cite

Humans and other tetrapods are considered to require apical-ectodermal-ridge, AER, cells for limb development, and AER-like cells are suggested to be re-formed to initiate limb regeneration. Paradoxically, the presence of AER in the axolotl, the primary regeneration model organism, remains controversial. Here, by leveraging a single-cell transcriptomics-based multi-species atlas, composed of axolotl, human, mouse, chicken, and frog cells, we first established that axolotls contain cells with AER characteristics. Surprisingly, further analyses and spatial transcriptomics revealed that axolotl limbs do not fully re-form AER cells during regeneration. Moreover, the axolotl mesoderm displays part of the AER machinery, revealing a novel program for limb (re)growth. These results clarify the debate about the axolotl AER and the extent to which the limb developmental program is recapitulated during regeneration.

show abstract

CRISPR/Cas9-based depletion of 16S ribosomal RNA improves library complexity of single-cell RNA-sequencing

Wang

Adler

2023

Preprint

View full text Add to dashboard Cite

Single-cell transcriptomics (scRNA-seq) has revolutionized our understanding of cell types and states in various contexts, such as development and disease. To selectively capture protein-coding polyadenylated transcripts, most methodologies rely on poly(A) enrichment to exclude ribosomal transcripts that constitute >80% of the transcriptome. However, it is common for ribosomal transcripts to sneak into the library, which can add significant background by flooding libraries with irrelevant sequences. The challenge of amplifying all RNA transcripts from a single cell has motivated the development of new technologies to optimize retrieval of transcripts of interest. This problem is especially striking in planarians, where a single 16S ribosomal transcript is widely enriched (20-80%) across single-cell methods. Therefore, we adapted the Depletion of Abundant Sequences by Hybridization (DASH) to the standard 10X scRNA-seq protocol. We designed single-guide RNAs tiling the 16S sequence for CRISPR-mediated degradation, and subsequently generated untreated and DASH-treated datasets from the same libraries to enable a side-by-side comparison of the effects of DASH. DASH specifically removes 16S sequences without off-target effects on other genes. By assessing the cell barcodes shared by both libraries, we find that DASH-treated cells have consistently higher complexity given the same amount of reads, which enables the detection of a rare cell cluster and more differentially expressed genes. In conclusion, DASH can be easily integrated into existing sequencing protocols and customized to deplete unwanted transcripts in any organism.

show abstract

In situ polyadenylation enables spatial mapping of the total transcriptome

Cited by 4 publications

References 50 publications

Extracellular matrix: Brick and mortar in the skeletal muscle stem cell niche

Extracellular matrix: Brick and mortar in the skeletal muscle stem cell niche

Multi-species atlas resolves an axolotl limb development and regeneration paradox

CRISPR/Cas9-based depletion of 16S ribosomal RNA improves library complexity of single-cell RNA-sequencing

Contact Info

Product

Resources

About