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GEO help: Mouse over screen elements for information. |
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| Status |
Public on Sep 05, 2024 |
| Title |
Gene expression library, day 2, Cynomolgus |
| Sample type |
SRA |
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| Source name |
56A1 cynomolgus induced pluripotent stem cells
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| Organism |
Macaca fascicularis |
| Characteristics |
cell line: 56A1 cynomolgus induced pluripotent stem cells
treatment: NPC differentiation and maintenance media
cell type: NPC differentiation
cell number_(cellranger): 1346
day: 2
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| Treatment protocol |
+/- 50 nM INK128
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| Growth protocol |
PSCs were singularized using Accutase (Sigma Aldrich) and counted on the Countess II FL Automated Cell Counter (Invitrogen). For human ESCs and cynomolgus iPSCs 1.25 x 105 cells/cm2 and for mouse EpiSCs 6.25 x 104 cells/cm2 were seeded onto Matrigel coated plates in UPPS media supplemented with 10 µM Y-27632 (R&D Systems) and incubated overnight, resulting in confluent cultures the following day for each species. For neural induction, media was exchanged to NPC differentiation and maintenance media consisting of 1:1 DMEM/F12 and Neuropan (PAN Biotech) supplemented with 0.5x N2 and B27 supplements, 1x MEM-NEAA, 1x GlutaMAX, 0.1 mM 2-Mercaptoethanol, 5 µg/mL Insulin, human recombinant (all Thermo Fisher Scientific), 10 µM SB431542 and 100 nM LDN193189 (both Peprotech). Cells were washed with Dulbecco’s phosphate buffered saline (DPBS; Thermo Fisher Scientific) before daily media exchange.
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| Extracted molecule |
polyA RNA |
| Extraction protocol |
Cell pellets were washed in PBS + 0.04% BSA and individual samples were labeled with a Cell Multiplexing Oligo (CMO) for sample demultiplexing in the analysis process. The labeling reaction was stopped by addition of PBS + 1% BSA. After washing, labeled samples from all species and conditions were pooled at equal ratios to obtain a suspension of 1.5 × 106 cells. Since neural differentiation sometimes leads to increased cell death and cell debris, the pooled samples were sorted with an FACS Aria Fusion (BD Biosciences) to remove small particles and cell clumps based on forward and side scatter (FSC and SSC) gating. Sorted cell pools were used at the appropriate volume for a target cell recovery of 12,000 for day 0 and 22,000 cells for days 2 and 4, and loaded onto a Chromium Next GEM Chip following the manufacturer’s instructions
cDNA was generated through a reverse transcription reaction inside GEMs. Two types of sequencing libraries were generated: A 3’ Gene Expression Library for transcriptome sequencing and a Multiplexing Library for CMO sequencing to demultiplex the pooled samples.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic single cell |
| Library selection |
cDNA |
| Instrument model |
Illumina NovaSeq X Plus |
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| Data processing |
3’ Gene Expression libraries of each time point (days 0, 2 and 4) were mapped against the mouse genome mm10 (GENCODE vM23/Ensembl98), the human genome GRCh38 (GENCODE v32/Ensembl98), both obtained from 10x Genomics, and the cynomolgus genome GCA_011100615.1 (Genome assembly 6.0), obtained from Ensembl. Mapping was performed using CellRanger 7.2.0 (10x Genomics). Using the cellranger multi function, sample demultiplexing was done in parallel to read alignment.
Assembly: mouse genome mm10 (GENCODE vM23/Ensembl98), the human genome GRCh38 (GENCODE v32/Ensembl98), both obtained from 10x Genomics, and the cynomolgus genome GCA_011100615.1 (Genome assembly 6.0)
Supplementary files format and content: For every demultiplexed sample: matrix.mtx: count matrix of genes and barcodes in Market Exchange Format (MEX); features.tsv: table of corresponding Ensemble Ids (first column) and Gene Ids (second column), corresponding to rows in matrix.mtx; barcodes.tsv: table of cell barcodes, corresponding to columns in matrix.mtx
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| Submission date |
Aug 23, 2024 |
| Last update date |
Sep 05, 2024 |
| Contact name |
Max Fernkorn |
| E-mail(s) |
m.fernkorn@lacdr.leidenuniv.nl
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| Organization name |
University Leiden
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| Department |
Leiden Academic Centre for Drug Research (LACDR)
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| Lab |
Drukker
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| Street address |
Einsteinweg 55
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| City |
Leiden |
| ZIP/Postal code |
2333 CC |
| Country |
Germany |
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| Platform ID |
GPL34842 |
| Series (2) |
| GSE275571 |
Single-cell multiome uncovers differences in glycogen metabolism underlying species-specific speed of development [mTORi] |
| GSE275572 |
Single-cell multiome uncovers differences in glycogen metabolism underlying species-specific speed of development |
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| Relations |
| BioSample |
SAMN43321969 |
| SRA |
SRX25813853 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM8479790_day2_cy_untr_barcodes.tsv.gz |
7.5 Kb |
(ftp)(http) |
TSV |
| GSM8479790_day2_cy_untr_features.tsv.gz |
192.6 Kb |
(ftp)(http) |
TSV |
| GSM8479790_day2_cy_untr_matrix.mtx.gz |
18.2 Mb |
(ftp)(http) |
MTX |
SRA Run Selector |
| Raw data are available in SRA |
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