GEO Accession viewer

NCBI > GEO > Accession Display

Not logged in | Login

GEO help: Mouse over screen elements for information.

Sample GSM6998162

Query DataSets for GSM6998162

Status

Public on Mar 31, 2023

Title

WT_stress_2a

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-Ty
condition: alkaline stress media, ambient CO2
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 either switched to alkaline-stress medium (RPMI 1640 supplemented with 1% IFS, 10 μg/mL gentamicin, 50 mM HEPES, and pH-adjusted to 8.1 with 10 N NaOH) under ambient CO2 or returned to standard conditions for an additional 48 h before harvest..

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.

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 file includes the following processed data, annotated as shown in brackets:
Supplementary files format and content: i) Log2(fold-change) in mRNA abundance in ∆bfd2 and wildtype parasites after treatment with alkaline stress [i.e. L2FC.∆bfd2_S.v.∆bfd2_US]
Supplementary files format and content: ii) Log2(fold-change) in mRNA abundance in ∆bfd2 as compared to its wildtype parebtal strain, under either stressed or unstressed conditions [i.e. L2FC.∆bfd2_S.v.WT_S]
Supplementary files format and content: iii) Corresponding negative log-transformed p-values for differential expression analysis [i.e. nL10p.∆bfd2_S.v.∆bfd2_US]
Supplementary files format and content: iv) Log-transformed FPKM values as genered within each comparison using the DeSeq2 fpkm() function [i.e. L10FPKM.WT_S].

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