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| Status |
Public on Aug 07, 2023 |
| Title |
16 R |
| Sample type |
SRA |
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| Source name |
P. falciparum infected RBCs
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| Organism |
Plasmodium falciparum |
| Characteristics |
cell type: Parasites
genotype: PfAP2-MRP knockout
time point: 16 hours post infection
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| Treatment protocol |
For 40 h.p.i. parasites, DMSO (control) or Rapamycin (to induce PfAP2-MRP knockout) was added to culture at 16 h.p.i. in the same cycle of sample collection. For 16 h.p.i. parasites, DMSO (control) or Rapamycin (to induce PfAP2-MRP knockout) was added to cultures at 35 h.p.i. in cycle 0 and parasites were collected at 16 h.p.i. in cycle 1.
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| Extracted molecule |
total RNA |
| Extraction protocol |
For 40 h.p.i. time point tight synchronous parasites were enriched using 63% Percol, washed twice with incomplete RPMI media and processed immediately on the 10X Chromium controller (10X Genomics, Pleasanton, CA). For 16 h.p.i. time point parasites were stained with Mitotracker Deep Red FM (Life Technologies, #M22426) for FACS analysis and flow sorting respectively. Briefly, 50 µl of SYBR Green I stained RBC’s were analyzed on BD LSR Fortessa Flow Cytometer with High Throughput sampler (BD Biosciences, San Jose, CA, USA) using BD FACS Diva Software v6.2 and 488 laser excitation / 530 emission filter to determine concentration of SYBR Green I positive cells per microliter. A BD Influx Cell Sorter (BD Biosciences, San Jose, CA, USA) with BD FACS Software v1.0.01 software was used to sort ~40,000 MitoTracker Deep Red FM - positive RBC’s using a 70 µm nozzle, a 640 nm laser excitation / 670 nm emission filter and a pressure setting of 30 psi. Post-sorted cell concentration and quality were checked using a Countess® II Automated Cell Counter (Invitrogen) and FLoid™ Cell Imaging Station (ThermoFisher). Finally, labelled cells (i.e. SYBR Green I or MitoTracker Deep Red FM positive cells) were then loaded onto a 10X chip (Chip G) and processed immediately on the 10X Chromium controller (10X Genomics, Pleasanton, CA).
Single cell libraries were constructed using the 10X Genomics Chromium Next GEM Single Cell 3ʹ Reagent Kits v3.1 with Single Index Kit T Set A. Due to the extremely low RNA content of single cell malaria parasite’s and the AT rich genome (~70% AT), modifications to the cDNA amplification and library preparation workflow were made accordingly. These modifications included - 30x cDNA amplification cycles; taking 50% cDNA as input into library generation; reducing fragmentation time to 2 minutes; and changing the extension time to 65 oC during index PCR. Individual library Qc was performed using the BioAnalyzer HS DNA Assay kit (Agilent).
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NovaSeq 6000 |
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| Description |
Rapamycin
Single Cell RNA-Seq (3' V3 Chemistry)
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| Data processing |
Cellranger.v5.0.1 standard pipeline was used for preprocessing and alignment.
The reads were aligned to the Hybrid genome of Human hg38 and P. falciparum pd37 v3 (PlasmoDB-46_Pfalciparum3D7_Genome.fasta) to remove any human transcript contamination. This was achieved using CellRanger standard pipeline using --nosecondary flag.
Genome_build: hg38, Pf3D7.v3
Supplementary_files_format_and_content: *.tar.gz: Tar archives include barcodes.tsv, features.tsv, and matrix.mtx files; raw counts.
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| Submission date |
Dec 15, 2021 |
| Last update date |
Aug 07, 2023 |
| Contact name |
AMIT KUMAR SUBUDHI |
| E-mail(s) |
amit.subudhi@kaust.edu.sa
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| Phone |
+96540375986
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| Organization name |
King Abdullah University of Science and Technology
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| Department |
BESE
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| Lab |
Pathogen Genomics
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| Street address |
Level4, Builiding, 2, KAUST
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| City |
Thuwal |
| State/province |
Thuwal |
| ZIP/Postal code |
239556900 |
| Country |
Saudi Arabia |
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| Platform ID |
GPL26836 |
| Series (2) |
| GSE190519 |
PfAP2-P DNA-binding protein is a master regulator of parasite pathogenesis during malaria parasite blood stages |
| GSE191025 |
PfAP2-P DNA-binding protein is a master regulator of parasite pathogenesis during malaria parasite blood stages (scRNA-seq) |
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| Relations |
| BioSample |
SAMN24108376 |
| SRA |
SRX13423634 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM5737644_16R_raw_counts.tar.gz |
26.9 Mb |
(ftp)(http) |
TAR |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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