2024-06-04
ENA first public
ERC000011
ENA-CHECKLIST
Wellcome Sanger Institute
INSDC center name
public
INSDC status
ERS19636955
SRA accession
E-MTAB-13071_2:WS_wEMB13942151
Submitter Id
17
week
age
ArrayExpress
broker name
not collected
collection date
human
common name
Protocols: All human developmental tissue samples used for this study were obtained from the MRC–Wellcome Trust-funded Human Developmental Biology Resource (HDBR; http://www.hdbr.org) with written consent and approval from the Newcastle and North Tyneside NHS Health Authority Joint Ethics Committee (08/H0906/21+5).All tissues were processed into single cell suspensions immediately upon receipt. Tissue was first minced in a tissue culture dish using scalpel. It was then digested with type IV collagenase (final concentration of 1.6 mg/ml; Worthington) in RPMI (Sigma-Aldrich) supplemented with 10% fetal bovine serum (FBS; Gibco), at 37°C for 30 min with intermittent agitation. Digested tissue was then passed through a 100-µm cell strainer and cells were pelleted by centrifugation at 500 g for 5 min at 4°C. Cells were then resuspended in 5 ml red blood cell lysis buffer (eBioscience) and left for 5-10 min at room temperature. It was then topped up with flow buffer (PBS containing 2% (v/v) FBS and 2 mM EDTA) to 45 ml prior to cell counting and antibody staining. Single cell suspensions were generated from 65 samples across yolk sac, liver, bone marrow, spleen, thymus, kidney and skin of 21 donors. The ages of the donors spanned from 4 pcw (post conception weeks) to 17 pcw.Dissociated cells were stained with anti-CD45 antibody and DAPI prior to sorting. Sorting by flow cytometry was performed with BD FACSAria Fusion Flow Cytometer. CD45 positive fraction was sorted from DAPI negative CD45 positive gate and CD45 negative fraction was sorted from DAPI negative CD45 negative gate. CD45 gating was contiguous so that no live cells were lost in sorting. For scRNA-seq experiments, Chromium single cell 3’ reagent kit from 10x Genomics were used. Unsorted, or DAPI-CD45+ or DAPI-CD45- FACS-isolated cells were loaded onto each channel of the Chromium chip following the manufacturer’s instructions before droplet encapsulation on the Chromium controller. Single-cell cDNA synthesis, amplification, gene expression (GEX) libraries were generated.
description
late embryonic stage
developmental stage
not collected
geographic location (country and/or sea)
Total
immunophenotype
F217
individual
not applicable
isolate
Homo sapiens
organism
Homo sapiens
organism
skin
organism part
Homo sapiens
scientific_name
female
sex
WS_wEMB13942151
title