Gene Expression Nebulas
A data portal of transcriptomic profiles analyzed by a unified pipeline across multiple species

Gene Expression Nebulas

A data portal of transcriptome profiles across multiple species

PRJNA471694: Single cell RNA-sequencing of human fetal kidneys

Source: NCBI / GSE114530
Submission Date: May 16 2018
Release Date: Feb 21 2019
Update Date: Mar 28 2019

Summary: 10X-based scRNA-seq data human fetal kidneys at 5 different ages

Overall Design: w9, w11, w13, w16 and w18 human fetal kidneys

GEN Datasets:
GEND000093
Strategy:
Species:
Tissue:
Healthy Condition:
Protocol
Growth Protocol: -
Treatment Protocol: -
Extract Protocol: One human embryo of w16 was isolated and the kidney dissected in cold saline solution (0.9% NaCl, Versylene Fresenius, FranceThe obtained kidney was decapsulated and kept on ice in dissociation buffer (DPBS + Penicillin 100U/ml + Streptomycin 0.1 mg/ml) before cutting it into 1 – 2 mm pieces. The pieces were washed 3 times with washing solution (Advanced DMEM F12 supplemented with: ITS commercial solution (Insulin – Transferrin – Selenium), Glutamax, Penicillin 100 U/ml and Streptomycin 0.1 mg/ml) with brief centrifugation (160g) in order to remove as many red blood cells as possible. The washed kidney tissue was then incubated with digestion solution (Trypsin/EDTA solution 0.25% and Collagenase-II 280 U/ml) and incubated overnight at 4°C. The next day, the digestion solution was removed, the kidney was rinsed with washing solution and incubated with washing solution for 30 min at 37°C with agitation. Subsequently, the sample was sequentially passed through sterile cell-strainers of 100, 70 and 40 µm pore size with the help of washing solution. The cells were then centrifuged, counted and viability was measured to be 78% (trypan blue assay) before proceeding with single-cell RNA sequencing library preparation.10X library: Single-cell RNA-seq libraries were prepared using the Chromium Single Cell 3' Reagent Kit, Version 2 Chemistry (10x Genomics) according to the manufacturer's protocol. Libraries were sequenced on a NextSeq500 in Mid Output mode using a version 2, 150 cycles kit (Illumina).; Human fetal embryos were isolated and the kidney dissected in cold saline solution (0.9% NaCl, Versylene Fresenius, France). The obtained kidneys were decapsulated and kept on ice in dissociation buffer (DPBS + Penicillin 100U/ml + Streptomycin 0.1 mg/ml) before cutting it into 1 – 2 mm pieces. The pieces were washed 3 times with washing solution (Advanced DMEM F12 supplemented with: ITS commercial solution (Insulin – Transferrin – Selenium), Glutamax, Penicillin 100 U/ml and Streptomycin 0.1 mg/ml) with brief centrifugation (160g) in order to remove as many red blood cells as possible. The washed kidney tissues were then incubated with digestion solution (Trypsin/EDTA solution 0.25% and Collagenase-II 280 U/ml) and incubated overnight at 4°C. The next day, the digestion solution was removed, the kidneys were rinsed with washing solution and incubated with washing solution for 30 min at 37°C with agitation. Subsequently, the samples were sequentially passed through sterile cell-strainers of 100, 70 and 40 µm pore size with the help of washing solution. The cells were then centrifuged, counted and viability was measured to be 78% (trypan blue assay) before proceeding with single-cell RNA sequencing library preparation.10X library: Single-cell RNA-seq libraries were prepared using the Chromium Single Cell 3' Reagent Kit, Version 2 Chemistry (10x Genomics) according to the manufacturer's protocol. Libraries were sequenced on a NextSeq500 in Mid Output mode using a version 2, 150 cycles kit (Illumina).
Library Construction Protocol: -
Sequencing
Molecule Type: poly(A)+ RNA
Library Source:
Library Layout: PAIRED
Library Strand: Forward
Platform: ILLUMINA
Instrument Model: Illumina NextSeq 500
Strand-Specific: Specific
Samples
Basic Information:
Sample Characteristic:
Biological Condition:
Experimental Variables:
Protocol:
Sequencing:
Assessing Quality:
Analysis:
Data Resource GEN Sample ID GEN Dataset ID Project ID BioProject ID Sample ID Sample Name BioSample ID Sample Accession Experiment Accession Release Date Submission Date Update Date Species Race Ethnicity Age Age Unit Gender Source Name Tissue Cell Type Cell Subtype Cell Line Disease Disease State Development Stage Mutation Phenotype Case Detail Control Detail Growth Protocol Treatment Protocol Extract Protocol Library Construction Protocol Molecule Type Library Layout Strand-Specific Library Strand Spike-In Strategy Platform Instrument Model Cell Number Reads Number Gbases AvgSpotLen1 AvgSpotLen2 Uniq Mapping Rate Multiple Mapping Rate Coverage Rate
Publications
Single-cell transcriptomics reveals gene expression dynamics of human fetal kidney development.
PLoS biology . 2019-02-21 [PMID: 30789893]