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| Status |
Public on Nov 07, 2023 |
| Title |
GC-JC-8721-2 |
| Sample type |
SRA |
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| Source name |
NC4
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| Organism |
Dictyostelium discoideum |
| Characteristics |
cell line: NC4
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| Treatment protocol |
For single cell RNAseq of a continuous developmental transition, we took a scrape of a feeding front of NC4 cells, from inside the bacterial zone through to the mound stage of development. Cells were immediately inoculated into ice-cold KK2 buffer (20mM KPO4, pH 6.0), and disaggregated by gentle pipetting. To remove the bacteria, cells were centrifuged at 720g for 2 minutes, then resuspended in ice-cold KK2.
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| Growth protocol |
Wild-type NC4 cells were cultured on lawns of Kelbsiella pneumoniae.
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| Extracted molecule |
total RNA |
| Extraction protocol |
Cell suspensions were loaded to the 10X Chromium Single Cell A Chip (PN-1000009) using the Chromium 3’ Library and Gel Bead Kit v2 (PN-120267) as described by the manufacturers (10X Genomics, California).
14 cycles of cDNA amplification were performed on the purified GEM-RT product, and cDNA was examined for quality using the Agilent 2200 Tapestation with the High-sensitivity D5000 screentape and reagents (Agilent Technologies, Waldbronn, Germany), and the Qubit 2.0 Fluorometer and Qubit dsDNA HS Assay Kit (Life Technologies, California, USA). 35 µL of cDNA was used to prepare the 10x 3’RNA library and 12 and 13 cycles were used for sample index PCR of replicates 1 and 2 respectively. Final cleaned libraries were quantified using the Qubit 2.0 Fluorometer and Qubit dsDNA HS Assay Kit and average fragment size checked using the Agilent D1000 screentape and reagents. The final library was run on a NextSeq500 Mid-output 150-cycle kit with a 26[8]98 cycle configuration to generate 130 million read pairs in total.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NextSeq 500 |
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| Description |
10x Genomics
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| Data processing |
Alignment, barcode counting, UMI counting, and filtering was performed by Cell Ranger v3.1.0 using default parameters. For the two biological replicates, totals of 2925 and 2404 single cell libraries passed the filter, with a median of around 15000 and 13000 transcripts per cell, and 2200 and 2300 genes per cell, for replicate one and two, respectively.
We excluded outliers with extremely high total UMI counts (1.5 interquartile ranges above the third quartile), cells with less than 3000 total UMI counts, and cells with less than 800 mapped genes. A total of 2671 and 2072 cells, from replicate one and two, respectively, were used for further analysis. Molecular counts of cells were normalised using size factors calculated with ‘scran’ package (Lun et al. Genome Biology, 2016). The processing steps were performed in R.
Supplementary files format and content: Tab-separated values files
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| Submission date |
Dec 06, 2022 |
| Last update date |
Nov 07, 2023 |
| Contact name |
Vlatka Antolovic |
| E-mail(s) |
v.antolovic@ucl.ac.uk
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| Organization name |
University College London
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| Department |
MRC Laboratory for Molecular Cell Biology
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| Street address |
Gower Street
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| City |
London |
| ZIP/Postal code |
WC1E 6BT |
| Country |
United Kingdom |
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| Platform ID |
GPL26360 |
| Series (1) |
| GSE220242 |
Collective signalling drives rapid jumping between cell states |
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| Relations |
| BioSample |
SAMN32077207 |
| SRA |
SRX18514262 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM6797147_Sample_2.csv.gz |
5.7 Mb |
(ftp)(http) |
CSV |
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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