|
| Status |
Public on Mar 31, 2023 |
| Title |
DD-BFD1∆bfd2_Shield_1a_rep2 |
| Sample type |
SRA |
| |
|
| Source name |
intracellular protozoan parasite, cultured in HFFs
|
| Organism |
Toxoplasma gondii |
| Characteristics |
strain: ME49
cell type: intracellular protozoan parasite, cultured in HFFs
genotype: ME49delta_ku80delta_bfd1::BFD1-Tydelta_hxgprt::pTUB1-DD-BFD1-Tydelta_bfd2::TdTomato
treatment: standard medium, +3uM Shield-1
replicate information: Biological replicate 2, Technical replicate 1
|
| Treatment protocol |
To establish infection, parasites were mechanically released from host cells by passage through a 27-gauge needle and innoculated onto confluent HFFs in standard medium.
After 4h, infected monolayers were washed twice with PBS, and the medium was replaced with new standard media containing either 3µM Shield-1 or an equivalent volume of vehicle. Experiments were harvested after an additional 48 hr of culture.
|
| Growth protocol |
For routine maintenance, parasites were cultured in human foreskin fibroblasts (HFFs) grown at 37°C under 5% CO2 in standard medium, consisting of DMEM (GIBCO) supplemented with 3% heat-inactivated fetal bovine serum (IFS), 2 mM L-glutamine, and 10 μg/mL gentamicin.
Passaging was performed on a 2-3 day cycle—prior to lysis of host cell monolayers—by scraping and transfer to fresh HFFs.
|
| Extracted molecule |
polyA RNA |
| Extraction protocol |
Parasites were isolated from host cells by syringe-release (described above) and filtering through a Millex filter unit (5 μm pore size, Merck Millipore) to remove host cell debris. RNA was isolated using the Rneasy mini plus kit (Qiagen).
Libraries for RNA-seq were prepared using the Swift Low-Input Library Prep Kit with poly(A) enrichment following manufacturer's protocols.
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| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NovaSeq 6000 |
| |
|
| Data processing |
Reads were trimmed to remove adapter sequences using Cutadapt v.3.6
Alignment to the T. gondii genome was performed with STAR v.2.7.0a.
Differential expression analysis was done using the DESeq2 package (v. 1.12.3) in R
Assembly: T. gondii ME49 (ToxoDB v.56 assembly)
Supplementary files format and content: xls files include i) relative mRNA abundance in BFD2-knockout parasites (DD-BFD1∆bfd2) relative to their parental strain (DD-BFD1) in both the presence and absence of Shield-1, and ii) within either strain, the relative change in mRNA abundance induced by treatment with Shield-1. FPKM values for each sample are also provided.
Supplementary files format and content: xls file includes the following processed data, annotated as indicated in brackets:
Supplementary files format and content: i) Log2(fold-change) in mRNA abundance in BFD2 knockouts (DDBFD1_dBFD2) relative to their parental strain (DDBFD1), under both Shield-1 (S1) and vehicle (V) treatment [i.e. L2FC.DDBFD1_dBFD2_S1.v.DDBFD1_S1]
Supplementary files format and content: ii) Log2(fold-change) in mRNA abundance either strain after treatment with Shield-1 [i.e. L2FC.DDBFD1_S1.v.DDBFD1_V]
Supplementary files format and content: iii) Corresponding negative log-transformed p-values for differential expression analysis [i.e. nL10p.DDBFD1_dBFD2_S1.v.DDBFD1_S1]
Supplementary files format and content: iv) Log-transformed FPKM values as genered within each comparison using the DeSeq2 fpkm() function [i.e. L10FPKM.DDBFD1_S1].
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| |
|
| Submission date |
Jan 27, 2023 |
| Last update date |
Mar 31, 2023 |
| Contact name |
Sebastian Lourido |
| E-mail(s) |
[email protected]
|
| Phone |
617-324-4920
|
| Organization name |
Whitehead Institute
|
| Lab |
Lourido
|
| Street address |
455 Main Street, Rm 555
|
| City |
Cambridge |
| State/province |
Massachusetts |
| ZIP/Postal code |
02142 |
| Country |
USA |
| |
|
| Platform ID |
GPL26742 |
| Series (1) |
| GSE223877 |
Effect of BFD2 (TGME49_311100) knockout on T. gondii gene expression during BFD1 induction |
|
| Relations |
| BioSample |
SAMN32941153 |
| SRA |
SRX19199858 |
