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Status |
Public on Aug 24, 2022 |
Title |
Gastruloid |
Sample type |
SRA |
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Source name |
Embryonic stem cells
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Organism |
Mus musculus |
Characteristics |
cell line: F1G4 condition: 28x pool of three different conditions gastruloid conditions: NN +Chi, NH+Chi, NH-Chi
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Growth protocol |
Male F1G4 mouse ES cells (George et al., 2007) were cultured on 6cm plates (Corning 430166) gelatinized with 0.1% gelatin (Sigma G1393) and coated with mitotically inactive primary mouse embryo fibroblasts (3-4x104 cells/cm²) with standard ES medium containing 15% FCS and 1000 U/ml leukemia inhibitory factor (LIF, Chemicon ESG1107) at 37°C and 5% CO2. ES cells were split every second day with a 1:10 dilution. For splitting, media was aspirated and cells were washed once with PBS and trypsinized (Trypsin-EDTA (0.05%) (Gibco 25300054)) for 5-10 minutes at 37°C. Trypsin was neutralized by 3 ml ES media and cells centrifuged for 5 minutes at 1000 rpm, after which the pellet was resuspended in ES media. ES cells were cultured in normoxia or hypoxia for 7 days. Gastruloids were then generated as described previously (Veenvliet et al., 2020) with some minor modifications. Briefly, ES cells were first depleted of feeders, then washed once in 5ml pre-heated (37°C) PBS containing MgCl2 and CaCl2 (Sigma D8662) and once in 5ml NDiff227 medium (Takara Y40002) pre-conditioned in normoxia or hypoxia. ES cells were then pelleted by centrifugation for 5 minutes at 1000rpm and resuspended in 250µl of NDiff227. 10µl of the cell suspension was mixed with 10µl of Trypanblue (Bio-Rad 1450021) for automated cell counting with Luna Automated Cell Counter. 400 live cells were plated in a volume of 35µl NDiff227 into each well of a 96-well round bottom, low attachment plate (Costar 7007 ultra-low attachment 96 well plate (7007)). Cells were then allowed to aggregate for 48 h under normoxic or hypoxic conditions. After 48h, the aggregates were treated with 3µM CHIR99021 (CHIR, Merck Millipore) in 150µl NDiff227 for 24h to induce robust gastruloid formation. For “-Chi” aggregates, 150µl NDiff227 without CHIR was added. Between 72 and 120h, medium was refreshed every 24h by removing 150µl of the old media and adding the same volume of new, pre-conditioned NDiff227.
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Extracted molecule |
polyA RNA |
Extraction protocol |
Gastruloids from NN+CHI, NH+CHI and NH-CHI conditions were generated as described above. From each condition, 28 structures were selected based on the occurrence of a symmetry breaking event and pooled for the single-cell experiment.. Gastruloids were picked with a p200 with the pipette tip cut-off at the 50µl mark, and pooled in a 1,5ml tube filled with ice cold PBS. The three pools were washed twice with ice cold PBS. Next, structures were dissociated in 50µl TrypLE Express (Gibco) for 20 minutes at 37ºC, with pipetting after 10 minutes. A 10x lipid-modified oligonucleotides (LMOs) – Barcode (BC) oligo solution was prepared (1:1; 2µM each) in PBS for each of the three samples on ice. From here on, every step has been performed on ice. After cell dissociation, LMOs – BC solutions were added to each sample separately (final concentration 200nM), and samples were incubated for 5min on ice. In the meantime a 10x co-anchor solution was prepared (2µM) in PBS. Following the 5 min incubation, co-anchor solution was added to each sample (final concentration 200nM), and samples were further incubated for 5min on ice. The reactions was then quenched by adding 1ml PBS+1% BSA to each tube. Cells were then washed twice with 1ml PBS+1% BSA with centrifugation steps performed for 5 minutes at 300g at 4°C in low DNAbind Eppendorf tubes. Next, cell pellets were resuspended in 100ul PBS+0,4% BSA and pooled together in one new tube. The cell suspension was filtered using Scienceware Flowmi Cell Strainers, 40µm. Cells where then centrifuged for 5 minutes at 300g at 4°C, and resuspended in 45ul PBS+0,4% BSA and The cell concentration was determined using a hemocytometer. Cells were then subjected to single-cell RNA sequencing (10x Genomics, Chromium™ Single Cell 3’ v3; one reaction). Single-cell libraries were generated according to the manual, with one modification: MultiSeq additive primer (5’-CTTGGCACCCGAGAATTCC-3’) was added at the cDNA amplification step (see McGinnis, Patterson et al 2019 for detailed protocol). During amplified cDNA cleanup, the MultiSeq BCs fraction was isolated and a separate library was performed as described in McGinnis, Patterson et al 2019. cDNA library was sequenced with a minimum of 400 million fragments according to parameters described in the manual and MultiSeq BCs library was sequenced with a minimum of 50 million fragments. Single-cell transcriptome profiling using MultiSeq
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Library strategy |
RNA-Seq |
Library source |
transcriptomic |
Library selection |
cDNA |
Instrument model |
Illumina NovaSeq 6000 |
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Data processing |
Illumina files were demultiplexed into FASTQ files using 10X Genomics' Single-Cell Gene Expression Software Cell Ranger ("mkfastq" pipeline, version 1.3.1, default parameters). FASTQ files were aligned, filtered, barcode and UMI counted using 10X Genomics' Single-Cell Gene Expression Software Cell Ranger ("count pipeline", version 1.3.1, default parameters The genomic reference "refdata-cellranger-mm10-1.2.0", prepared by 10x Genomic was used to alignment. To de-multiplex samples within our single scRNAseq dataset, we used the deMULTIplex R package (version 1.0.2). The initial quality control was performed with Seurat. Cells with less than 3 cells, less than 200 or more than 2,500 unique feature counts and a mitochondrial fraction above 5% were removed from the analysis. Quality control features were checked for each individual sample. Genome_build: mm10
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Submission date |
Jan 27, 2022 |
Last update date |
Aug 24, 2022 |
Contact name |
Natalia Lopez-Anguita |
E-mail(s) |
anguita@molgen.mpg.de
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Organization name |
Max Planck Institute for Molecular Genetics
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Department |
Genome Regulation
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Lab |
Bulut-Karslioglu Lab
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Street address |
Ihnestraße 63
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City |
Berlin |
State/province |
Berlin |
ZIP/Postal code |
14195 |
Country |
Germany |
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Platform ID |
GPL24247 |
Series (2) |
GSE178628 |
Hypoxia induces an early primitive streak signature, enhancing spontaneous elongation and lineage representation in gastruloids |
GSE195544 |
Hypoxia induces an early primitive streak signature, enhancing spontaneous elongation and lineage representation in gastruloids |
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Relations |
BioSample |
SAMN25331598 |
SRA |
SRX13946817 |
Supplementary file |
Size |
Download |
File type/resource |
GSM5840322_barcodes.tsv.gz |
44.8 Kb |
(ftp)(http) |
TSV |
GSM5840322_features.tsv.gz |
284.1 Kb |
(ftp)(http) |
TSV |
GSM5840322_matrix.mtx.gz |
34.4 Mb |
(ftp)(http) |
MTX |
SRA Run Selector |
Raw data are available in SRA |
Processed data provided as supplementary file |
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