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GEO help: Mouse over screen elements for information. |
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| Status |
Public on Mar 17, 2023 |
| Title |
run3_D1_iX01 |
| Sample type |
SRA |
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| Source name |
R1
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| Organism |
Mus musculus |
| Characteristics |
cell line: R1
treatment: control
cell type: ES cell
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| Treatment protocol |
We used ML-792, a highly selective inhibitor of the SUMO E1 enzyme, to decrease the global level of SUMOylation in mouse ESCs, with two rounds of 48 h treatments (2.5mM) between D1 and D3, D8 and D10
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| Growth protocol |
Serum+Lif (D1,D3,D8,D10). N2B27 + Lif (D18, ESC_N2B27Lif, Gastruloid_AgW, ESC_AgW, Gastruloid_F2, Gastruloid_F5, ELS_M7)
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| Extracted molecule |
total RNA |
| Extraction protocol |
Cells were sorted into 384-well cell capture plates using a BD FACSAria III Cell Sorter (BD Biosciences) to collect live cells and sort only singlets. Plates were snap frozen on dry ice and stored at -80°C until further processing. All single cell libraries were prepared with the same conditions and reagents using the MARS-seq protocol as previously described (Jaitin et al, Science, 2014).
All single cell libraries were prepared with the same conditions and reagents using the MARS-seq protocol as previously described (Jaitin et al, Science, 2014). Briefly, a Bravo Automated Liquid Handling Platform (Agilent) was used to reverse transcribe (Invitrogen #18080085) mRNA into cDNA with an oligonucleotide containing both the unique molecule identifiers (UMIs) and cell barcodes. Unused oligonucleotides were removed by Exonuclease I (New England Biolabs #M0293S) treatment. cDNAs were pooled (each pool containing half of a 384-well plate) for second strand synthesis (New England Biolabs #E6111S) and in vitro transcription amplification (New England Biolabs #E2040S). DNA template was removed (Invitrogen #AM2238) before fragmenting (Invitrogen #AM8740) and ligating (New England Biolabs #M0204S) resulting RNA to an oligo containing the pool barcode and Illumina sequences. Finally, RNA was reverse transcribed (Agilent Technologies #600107) and libraries were amplified (Roche #7958935001). Libraries were quantified with a Qubit 2.0 (Invitrogen) and their size distribution was determined by a 4200 TapeStation System (Agilent Technologies). Finally, libraries were pooled at equimolar concentration and sequenced on an Illumina NextSeq500, in 8 sequencing runs, using high-output 75 cycles v2.5 kits (Illumina #20024906).
ScRNA-seq (MARS-seq)
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NextSeq 500 |
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| Description |
counts_run3.csv.gz
meta_run3.csv
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| Data processing |
We used the MARS-seq2.0 pipeline, the reads structure is as follow : R1 : 5I.4P.66M (ignore 5 first bases, followed by 4 nt of plate barcode and finally the 66 nt of the insert), R2 : 7W.8R.1I (7 nt of well barcodes, 8nt of UMIs and 1 nt ignore)
Genome_build: mm10
Supplementary_files_format_and_content: raw counts tables and metadata files (including FACS output)
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| Submission date |
Jul 22, 2021 |
| Last update date |
Mar 17, 2023 |
| Contact name |
Yann Loe-Mie |
| E-mail(s) |
Yann.loe-mie@pasteur.fr
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| Organization name |
Institut Pasteur
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| Department |
Computational Biology
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| Lab |
Bioinformatics and Biostatistics HUB
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| Street address |
25 rue du Dr Roux
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| City |
Paris |
| ZIP/Postal code |
75015 |
| Country |
France |
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| Platform ID |
GPL19057 |
| Series (2) |
| GSE180489 |
HypoSUMOylation in embryonic stem cells generates head-and-trunk embryo-like structure |
| GSE180679 |
HypoSUMOylation in embryonic stem cells generate head-trunk embryo-like structures [scRNAseq] |
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| Relations |
| BioSample |
SAMN20352100 |
| SRA |
SRX11524271 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
| Processed data are available on Series record |
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