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Single Cell RNA-sequencing of murine back skin interfollicular epidermis basal cells

ABSTRACT: Single Cell RNA-sequencing of murine back skin interfollicular epidermis basal cells

PROVIDER: PRJNA611558 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR11271748.fastq.gz	Fastqsanger.gz
	SRR11271748_1.fastq.gz	Fastqsanger.gz
	SRR11271748_2.fastq.gz	Fastqsanger.gz
	SRR11271749_1.fastq.gz	Fastqsanger.gz
	SRR11271749_2.fastq.gz	Fastqsanger.gz

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Project description:The skin interfollicular epidermis (IFE) is the first barrier against the external environment and its maintenance is critical for survival. Two seemingly opposite theories have been proposed to explain IFE homeostasis. One posits that IFE is maintained by a long-lived slow-cycling stem cell (SC) population that give rise to short-lived transit-amplifying (TA) cell progeny, while the other suggests that homeostasis is achieved by a single committed progenitor (CP) that balances stochastic fate. Here, we probed the cellular heterogeneity within the IFE using two different inducible CREER targeting IFE progenitors. Quantitative analysis of clonal fate data and proliferation dynamics demonstrate the existence of two distinct proliferative cell compartments composed of slow-cycling SC and CP, both of which undergo population asymmetric self-renewal. However, following wounding, only SCs contribute substantially to the repair and long-term regeneration of the tissue, while CP cells make a minimal and transient contribution. Transcriptional profile of InvCREER/RosaYFP and K14CREER/RosaYFP targeted FACS-isolated alpha6 integrin-high CD34-neg basal cells from mouse tail epidermis. Skin epidermis was removed from tail bone and incubated overnight in HBSS (Gibco) 0.25% trypsin (Gibco) at 4M-BM-0C. Epidermis was separated from the dermis and incubated on a rocking plate (100 rpm) at room temperature for 5 min. Basal cells were mechanically separated from the epidermis by flushing 10 times under the epidermis. Tissues were then cut in pieces of 1 mm2 with scalpel, and trypsin was neutralized by adding DMEM medium (Gibco) supplemented with 2% Chelex Fetal Calf Serum (FCS). Samples were filtrated on 70 and 40M-BM-5m filter (Falcon). Immunostaining was performed using biotin-conjugated anti-CD34 (clone RAM34; BD Biosciences) PE-conjugated anti-M-NM-16-integrin (clone GoH3; BD biosciences). Primary antibodies were washed with 2% FCS/PBS and cells were incubated for 30 min in APC conjugated streptavidin (BD Biosciences) secondary antibodies, on ice, with shaking every 10 min. Living K14- and involucrin-expressing epidermal cells were gated by forward scatter, side scatter, negative staining for Hoechst dye and by following the YFP signal. Basal cells from the interfollicular epidermis were targeted using CD34 negative alpha 6 high gating. Fluorescence-activated cell sorting analysis was performed using FACSAria I at high pressure (70 psi) and FACSDiva software (BD Biosciences). Sorted cells were harvested directly in the lysis buffer provided by the RNeasy microkit (QIAGEN) supplemented with 1M-BM-5l of beta-mercaptoethanol for every 100M-BM-5l of lysis buffer. RNA extraction was performed on freshly sorted cells according to the manufacturerM-bM-^@M-^Ys protocol.

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Single Cell RNA-sequencing of murine back skin interfollicular epidermis basal cells

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