2025-07-01
ENA first public
ERC000011
ENA-CHECKLIST
The University of Manchester
INSDC center name
public
INSDC status
ERS20598191
SRA accession
E-MTAB-14249:Sample 3
Submitter Id
ArrayExpress
broker name
HES3
cell_line
cardiac progenitor
cell_type
not collected
collection date
human
common name
Protocols: Differentiations were started on sequential days so the different timepoints could be collected and processed on the same day. Sample 1 was collected on day 3 and day 3.75 (50:50 mix); Samples 2, 3 and 4 were collected on days 4.75, 5.75 and 12 respectively. Embryoid bodies were dissociated to single cells using TrypLE Select.  Gene expression libraries were prepared from the single cells using the Chromium Controller and Single Cell 3ʹ Reagent Kits v3 (10x Genomics, Inc. Pleasanton, USA) according to the manufacturer’s protocol (CG000183 Rev A). Briefly, nanoliter-scale Gel Beads-in-emulsion (GEMs) were generated by combining barcoded Gel Beads, a master mix containing cells, and partitioning oil onto a Chromium chip. Cells were delivered at a limiting dilution, such that the majority (90-99%) of generated GEMs contain no cell, while the remainder largely contain a single cell. The Gel Beads were then dissolved, primers released, and any co-partitioned cells lysed. Primers containing an Illumina TruSeq Read 1 sequencing primer, a 16-nucleotide 10x Barcode, a 10-nucleotide unique molecular identifier (UMI) and a 30-nucleotide poly(dT) sequence were then mixed with the cell lysate and a master mix containing reverse transcription (RT) reagents. Incubation of the GEMs then yielded barcoded cDNA from poly-adenylated mRNA. Following incubation, GEMs were broken, and pooled fractions recovered. First-strand cDNA was then purified from the post GEM-RT reaction mixture using silane magnetic beads and amplified via PCR to generate sufficient mass for library construction. Enzymatic fragmentation and size selection were then used to optimize the cDNA amplicon size. Illumina P5 & P7 sequences, a sample index, and TruSeq Read 2 sequence were added via end repair, A-tailing, adaptor ligation, and PCR to yield final Illumina-compatible sequencing libraries.
description
not collected
geographic location (country and/or sea)
not applicable
isolate
Homo sapiens
organism
Homo sapiens
organism
embryonic stem cell
progenitor cell type
Homo sapiens
scientific_name
Sample 3
title