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Sample GSM6957577

Query DataSets for GSM6957577

Status

Public on May 12, 2023

Title

PB52-T6-Rep-1

Sample type

SRA

Source name

Plasmodium falciparum

Organism

Plasmodium falciparum

Characteristics

strain: PB52 (MRST mutant)
genotype: piggyBac mutant
disrupted gene id: PF3D7_1136600
developmental stage: early ring

Treatment protocol

Cultures of wildtype NF54 and PB52 (MRST mutant) were synchronized at ~2% ring stage with 10% sorbitol solution. After 48 hours of growth, cultures were again synchronized with 10% sorbitol. After 8hrs, cultures were synchronized for a third time with 10% sorbitol to yield highly synchronized cultures. 0hpi was determined (half schizonts and half ring cultures) and samples were collected for RNA extraction and sequencing at 6hpi, 12hpi, 24hpi, 36hpi, and 48hpi.

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 0.01 mg/ml gentamicin, 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 cold 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

NextSeq 550

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
Assembly: P. falciparum reference genome (PlasmoDB version 47)
Supplementary files format and content: feature_counts_output_MRST.xlsx is the output of Feature counts and contains ID (GeneID), chromosome (Chr), and the counts (number of reads), for each mapped gene from each RNAseq sample.

Submission date

Jan 23, 2023

Last update date

May 12, 2023

Contact name

Caroline Simmons

E-mail(s)

[email protected]

Organization name

University of South Florida

Street address

3720 E Spectrum Blvd