2024-04-30
ENA first public
ERC000011
ENA-CHECKLIST
SAMEA114541669
External Id
UOM
INSDC center name
2023-10-26T14:03:56Z
INSDC last update
public
INSDC status
ERS16522998
SRA accession
E-MTAB-13500:WT
Submitter Id
25
day
age
ArrayExpress
broker name
Man-13 hESC
cell_line
not collected
collection date
human
common name
Protocols: Day 25 organoids were washed twice with PBS and three organoids were pooled together per sample and resuspended in 1 mL of TrypLETMExpress in 1.5 mL centrifuge tubes. Samples were incubated at 37°C for 15 minutes with occasional flicking and then passed through a 27G needle (BD Biosciences, #305540) to dissociate organoids to a single cell suspension. The single cell suspension was then centrifuged at 400xg for 3 minutes, the supernatant was discarded, and cells resuspended in 1 mL of basal medium. Cells were counted and diluted to 1000 cells per μL, 16 μL of the diluted cell suspension and 30.6 μL of nuclease-free water were used per reaction. The cells were passed through the 10x Chromium (10x Genomics) system as indicated on the manufacturer’s protocol (CG000315 Rev B). Gene expression libraries were prepared from single cells using the Chromium Controller and Single Cell 3ʹ Reagent Kits v3.1 (10x Genomics, Inc. Pleasanton, USA). 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 cells, 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
normal
disease
HNF1B wild type
genotype
not collected
geographic location (country and/or sea)
pool of 3 organoids
individual
not applicable
isolate
Homo sapiens
organism
Homo sapiens
organism
kidney organoid
organism part
embryonic stem cell
progenitor cell type
Homo sapiens
scientific_name
male
sex
WT
title