Homo sapiens
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.
ENA first public
2024-04-30
External Id
SAMEA114541669
INSDC center name
UOM
INSDC last update
2023-10-26T14:03:56Z
INSDC status
public
Submitter Id
E-MTAB-13500:WT
age
25
broker name
ArrayExpress
cell_line
Man-13 hESC
collection date
not collected
common name
human
disease
normal
genotype
HNF1B wild type
geographic location (country and/or sea)
not collected
individual
pool of 3 organoids
isolate
not applicable
organism part
kidney organoid
progenitor cell type
embryonic stem cell
sample name
E-MTAB-13500:WT
scientific_name
Homo sapiens
sex
male