GSM3855911: 72 hpf, periderm, replicate A; Danio rerio; RNA-Seq - SRA

SRX5982370: GSM3855911: 72 hpf, periderm, replicate A; Danio rerio; RNA-Seq
1 ILLUMINA (Illumina HiSeq 2000) run: 24.6M spots, 1.3G bases, 821.3Mb downloads

Submitted by: NCBI (GEO)

Study: Tissue-specific transcriptomes reveal gene expression trajectories in two maturing skin epithelial layers in zebrafish embryos

PRJNA547573 • SRP200656 • All experiments • All runs

show Abstracthide Abstract

We purified (FACS) and profiled (RNA-Seq) zebrafish embryo skin cells vs. nonskin cells at three early developmental stages. At two of the stages, we further purified and profiled two distinct epithelial layers of the skin: the periderm and basal cells. Our comprehensive expression analysis provides the first reported transcriptomes of these two cell types, reveals gene expression dynamics in space and time, and identifies common and distinct features of epithelial maturation in the two layers. Given the conserved nature of skin development, we believe our study to be of interest to those studying skin in other vertebrates. Overall design: At each of 20 SS, 52 hpf, and 72 hpf, we used krt4:dsRed transgenic zebrafish embryos and FACS to prepare and run two RNA-Seq conditions: all skin vs. nonskin1, with two replicates for each condition. At each of 52 hpf and 72 hpf, we used krt4:dsRed;krt5:GFP transenic zebrafish embryos and FACS to prepare and run three RNA-Seq conditions: periderm vs. basal cells vs. nonskin2, with two replicates for each condition, except 52 hpf nonskin2 has only one replicate.

Sample: 72 hpf, periderm, replicate A

SAMN11969615 • SRS4887875 • All experiments • All runs

Organism: Danio rerio

Library:

Instrument: Illumina HiSeq 2000

Strategy: RNA-Seq

Source: TRANSCRIPTOMIC

Selection: cDNA

Layout: SINGLE

Construction protocol: Dechorionated embryos were transferred into 1.5 ml tubes and rinsed with Ca2+-free Ringer's solution for 15 min. During this incubation, yolk was removed by gently pipetting embryos through a 200 µl tip (with the end cut off) three to five times. Yolk-free embryos were transferred into a 35 mm petri dish with 5 ml of 0.25% Trypsin-EDTA. Embryos were incubated at 28.5 °C and homogenized with a 200 µl tip every 10 min until most cells were dissociated (20 to 50 min). 55 µl 100 mM CaCl2 and 550 µl FCS were added to stop digestion. Dissociated cells were transferred into a 15 ml tube and centrifuged at 300 g for 5 min at 15 °C. Cells were rinsed once with 10 ml suspension solution (colorless Leibovitz medium L-15 with 0.3 g/L glutamine, 0.8 mM CaCl2, Pen 50U/ml, Strep 0.05 mg/ml, and 1% FCS). Dissociated cells were resuspended in suspension solution to ~10^7 cells/ml and immediately proceeded to cell sorting at the UCLA Broad Stem Cell Research Center Flow Cytometry Core. BD FACS ARIA II SORP instruments sorted cells, using a 488 nm laser for GFP detection and a 561 nm laser for RFP (dsRed) detection. Sorted cells in suspension solution were immediately lysed using the Qiagen RNeasy kit. Lysis was completed within two hours of dissociating cells. Total RNA was isolated following the Qiagen RNeasy kit and stored at –80 °C. Quality of all samples was assayed with an Agilent Bioanalyzer, and only samples with RNA Integrity Number > 8 were used for creating sequencing libraries. Poly-A bead-purified RNA-Seq libraries were prepared with the Illumina TruSeq RNA Library Prep Kit.

Experiment attributes:

GEO Accession: GSM3855911

Links:

NCBI link: NCBI Entrez (gds)

Runs: 1 run, 24.6M spots, 1.3G bases, 821.3Mb

Run	# of Spots	# of Bases	Size	Published
SRR9211503	24,600,526	1.3G	821.3Mb	2019-08-24

ID:: 8018676

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