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Sample GSM6965729

Query DataSets for GSM6965729

Status

Public on Mar 31, 2023

Title

HA-BFD2_stress_IP

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_bfd2::HA-BFD2
treatment: alkaline stress
sample type: IP (enriched by HA-BFD2 pull-down)

Treatment protocol

Extracellular parasites were innoculated onto confluent HFFs and allowed to invade in standard medium. After 4 hr, monolayers were washed twice with PBS and 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) to induce differentiation, then left for an additional 72hr 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 by scraping the infected monolayers and transfering to fresh HFFs.

Extracted molecule

polyA RNA

Extraction protocol

At the time of harvest, monolayers were scraped and serially extruded through 27-and 30-gauge needles to release intracellular parasites. Parasites were then isolated from host cell debris by additional passaging through a Millex filter unit (5 μm pore size, Merck Millipore), pelleted by centrifugation, and resuspended in MAGNA-RIP lysis buffer. For an 'input' sample, 10% of the starting lysate was set aside. The remaining 90% was subjected to IP with mouse anti-HA antibodies (clone 16B12, Abcam) using the MAGNA-RIP kit (Millipore) as described in the product technical manual. Total RNA was isolated in parallel from both enriched (IP) and unenriched (input) samples by canonical phenol:chloroform extraction.
Libraries for RNA-seq were prepared using the Swift Low-Input Library Prep Kit with poly(A) enrichment following manufacturer's protocols.

Library strategy

RIP-Seq

Library source

transcriptomic

Library selection

other

Instrument model

Illumina NovaSeq 6000

Data processing

Reads were trimmed to remove adapter sequences with Cutadapt (v.3.6) and aligned to the T. gondii genome with STAR (v.2.7.0a). BFD2-bound transcripts were identified based on the following enrichment analysis pipeline:
i) From raw read counts, we calculated a read coverage score for each message, defined as the square root of the sum of squares for reads in both the IP and input control. Only transcripts with i) read coverage greater than 10 and ii) more than 5 mapped reads in both the IP and input were included in downstream analysis.
ii) Within each trimmed data set (i.e. IP and input), raw reads for each gene were convered to Transcripts Per Million (TPM), normalizing for sequencing depth.
iii) An enrichment ratio was generated for each gene, defined as TPM in the IP divided by TPM in the input.
iv) Gaussian mixture modeling was perfomred on log2-transformed enrichment ratios, which fit two Gaussian curves to the data—an upper 'enriched' distribution which comprised only the highest enrichment ratios and a lower 'background' distribution corresponding to all other unenriched messages.
v) Posterior probabilities were generated from this model and used to calculate an odds ratio for each gene. To set a cutoff for calling BFD2-enriched transcripts, we used the log of the odds (LOD) ratio between the two Gaussian functions. LOD greater than 0 indicates a higher probability of falling within the upper distribution compared to lower background distribution; thus, LOD > 0 was used as a threshold to call individual transcripts as highly enriched.
Assembly: T. gondii ME49 (ToxoDB v.56 assembly)
Supplementary files format and content: Tabulated xls file includes raw read counts for all genes measured in both IP and input samples (Tab "RIP_ALL") and the results of each step in the analysis pipeline outlined above (Tab "RIP_TRIMMED"), performed on a reduced data set trimmed to only include only genes meeting our minimal threshold for detection.

Submission date

Jan 24, 2023

Last update date

Mar 31, 2023

Contact name

Sebastian Lourido

E-mail(s)

[email protected]

Phone

617-324-4920

Organization name

Whitehead Institute