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Sample GSM5685092

Query DataSets for GSM5685092

Status

Public on Jan 31, 2022

Title

pb104-rep2-BTZ-T1

Sample type

SRA

Source name

pb104_BTZ-T1

Organism

Plasmodium falciparum

Characteristics

strain: PB104
genotype: piggyBac mutant
disrupted gene id: PF3D7_0104200
drug treatment: Bortezomib (BTZ) exposure

Treatment protocol

Cultures of wildtype NF54 and pB- mutant pB104 were highly synchronous rings, then split equally into two control and two experiments T75 flasks each. T0 samples were harvested. All parasites were grown at the normal human body temperature (37C) to early ring-stage. Two flasks of were then exposed to 3-cycles growth of low-dose of each antimalarial drug (dihydroartemisin-DHA and Bortezomib-BTZ), while the remaining two flasks without any drug exposure. After the third cycles-growth (T1), RNA was harvested simultaneously from all conditions for RNAseq.

Growth protocol

Parasite cultures were maintained according to standard methods with gassing (5% O2 and 5% CO2, nitrogen balanced) and 5% hematocrit (O+ blood; Interstate Blood Bank) in RPMI 1640 medium (Invitrogen) supplemented with 0.5% Albumax II (Invitrogen), 0.25% sodium bicarbonate, and maintained at 37°C.

Extracted molecule

total RNA

Extraction protocol

Parasites were separated from red blood cells via incubation with 0.015% saponin at room temperature for 5 min. Cultures were then pelleted and washed three times in 10 mL room temperature PBS. Samples were stored at − 80 °C in 1 mL TRIzol reagent (Fisher Scientific, Hampton, NH) until extraction. At RNA extraction, 200 μl of chloroform was added and samples were vortexed vigorously for 15 seconds, followed by incubation at room temperature for up to 5 minutes. Samples were then spun down at 12000×g (10,800 rpm) at 4 °C for 10 minutes and the supernatant discarded. 1 mL of 75% ethanol was added to the pellet, and samples were spun down at 10000×g (9800 rpm) for 5 minutes. The resulting supernatant was discarded and the pellet briefly allowed to dry. The RNA pellet was then dissolved in 20–50 μl of DEPC-treated water while being incubated at 55 °C for 10–15 min.
0.5 μg–1.0 μg of RNA samples were prepped for sequencing using the Illumina TruSeq Stranded mRNA Kit as per kit protocol. Library quantification was measured by qPCR and TapeStation (Agilent Technologies). Sequencing was performed on an Illumina NextSeq V2.5 mid-output 300-cycle.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina NextSeq 500

Data processing

RNAseq reads from each sample were aligned to the P. falciparum reference genome usinf HISAT2.
Mapped reads were used to assemble known transcripts from the reference and their abundances were estimated using SAMtools and the abundances were estimated using Feature Counts
Genome_build: P. falciparum reference genome (PlasmoDB version 47)
Supplementary_files_format_and_content: RNAseq_data_processed_counts.txt is the output of Fecture counts and contains ID (Geneid), chromosome (Chr),vcoordenadenades (Start and End), Strand (+ or -), Length and rhe counts (number of reads), for each mapped gene from each RNAseq sample.

Submission date

Nov 10, 2021

Last update date

Jan 31, 2022

Contact name

Camilla Valente Pires

E-mail(s)

camilla@usf.edu

Organization name

University of South Florida

Street address

3720 Spectrum Blvd, 404