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Sample GSM5842369

Query DataSets for GSM5842369

Status

Public on Jan 31, 2022

Title

3D7_KO

Sample type

SRA

Source name

Red blood cells infected P. falciparum

Organism

Plasmodium falciparum 3D7

Characteristics

strain: 3D7
age: 28-32h after cycle initiation
genotype: knock-out of PfMRP1 transporter

Treatment protocol

Parasites were tightly synchronized with 5% sorbitol in intervals of 20 hours for 3 consecutive life cycles.

Growth protocol

P. falciparum 3D7 and Dd2 strains (MRA-120 and MRA-156, MR4-Malaria Resources) were maintained at 4% hematocrit in RBCs with RPMI-1640 (Gibco) supplemented with 2 g/L sodium bicarbonate, 2 mM L-glutamine, 25 mM HEPES (Gibco), 25 µg/mL gentamycin (Gibco), 100 µM hypoxanthine (Sigma-Aldrich) and 0.25% (w/v) AlbuMAXII (Gibco) with daily medium changes. Parasites were maintained at 37 °C in an airtight environment with 3% O2, 5% CO2 and 92% of N2.

Extracted molecule

total RNA

Extraction protocol

RNA was extracted from trophozoite stages, estimated between 28 to 32 hours old, using a RNeasy Mini Kit (Qiagen)
Libraries and sequencing were outsourced (Macrogen Inc), using TruSeq RNA Illumina Library v2 construction (nonstranded polyA enrichment) and HiSeq2500 Illumina 2x100bp 2Gb (20M pair-end reads) throughput-based sequencing.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiSeq 2500

Data processing

Transcriptome datasets for both 3D7 and Dd2 were mapped against the 3D7 v3 (Böhme, U. et al., Wellcome Open Res., 2019) reference sequence using HISAT2 2-2.0.0-beta (Kim, D. et al., Nat.Biotechnol, 2019) (--rna-strandness RF --max-intronlen 5000).
Read counts were determined using HTSEQ v0.6.0 (Anders, S. et al., Oxf. Engl., 2015) (-r pos -s no). Deconvolution analysis was performed as described previously in Aunin, E. et al., PLOS Pathogens, 2020.
Differentially expressed genes were determined using edgeR (Robinson, M.D. et al., Bioinforma, 2010), with genes expressed at <=3 counts per million (cpm) excluded.
Because there were no biological replicates for any condition, we used the exact test with dispersion set to 0.01, as suggested for data concerning genetically identical model organisms (https://www.bioconductor.org/packages/release/bioc/vignettes/edgeR/inst/doc/edgeRUsersGuide.pdf).
We used a false discovery rate (FDR) cut off of 0.01 and a fold change cut off of 2 to call differentially expressed genes.
Genome_build: 3D7 v3 reference sequence (Böhme, U. et al., Wellcome Open Res., 2019)
Supplementary_files_format_and_content: Table with genes up or down in KO relative to WT with FDR <= 0.01 and fold change > 2. Direction of up or downregulation, gene ID, false discovery rate (FDR), log counts per million (logCPM), log fold change (logFC), and gene description (Desc).

Submission date

Jan 28, 2022

Last update date

Jan 31, 2022

Contact name

Nuno S. Osório

E-mail(s)

nosorio@med.uminho.pt

Organization name

ICVS

Street address

Campus de Gualtar