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Sample GSM4099292

Query DataSets for GSM4099292

Status

Public on Jan 20, 2020

Title

HT 24 hpi, rep A

Sample type

SRA

Source name

P. acanthamoebae UV-7

Organism

Parachlamydia acanthamoebae UV-7

Characteristics

strain: UV-7
developmental stage of endosymbiont: 24 hr post infection
multiplicity of infection: 100

Treatment protocol

At respective time points post infection infected amoebae were harvested, resuspended in sucrose buffer (35 mM Tris-HCl, 250 mM sucrose, 25 mM KCl, 10 mM MgCl2), supplemented with 50 µg/ml Rifampicin, and then disrupted by 1 min vortexing in the presence of glass beads. Suspensions were filtered through 5 µm filters, and bacteria were collected by 2 min high-speed centrifugation. Extracellular bacteria were pelleted, resuspended in sucrose buffer and treated like the other two time points.

Growth protocol

Amoeba-endosymbiont cultures, originating from the same ancestral population, were maintained at 20°C in TSY (30 g/L trypticase soy broth, Oxoid, 10 g/L yeast extract; pH 7.3) for 14 months under two different experimental set-ups. In one, HT was the predominant mode of transmission, whereas in the other VT was the main mode of transmission. For the RNA-Seq infection experiment, uninfected amoebae were seeded at low density 3 days prior to infection and incubated at 20°C, before purified endosymbionts were added. These originated from the last passage of amoeba-endosymbiont cultures from the HT and VT set-ups, and extracellular endosymbionts were harvested from the medium supernatant after one week of incubation. Two hours after infection, cultures were washed 3x and infected cultures were harvested (2 hpi time point) or further incubated at 20°C in TSY for 24 hrs. Released EBs were collected 1 week after infection as the final time point.

Extracted molecule

total RNA

Extraction protocol

After collection of cells, pellets were immediately resuspended in TRIzol reagent, followed by bead-beating (30s, 4.5 m/s, lysing matrix A tubes, MP Biomedicals). Subsequent RNA extraction was performed according to the TRIzol guidelines. Residual DNA was removed using the Turbo DNA-free Kit (Thermo Fisher Scientific).
The sequencing facility (Vienna BioCenter Core Facilities) removed ribosomal RNA using Illumina's Ribo-Zero Gold rRNA Removal Kit (Epidemiology). RNA libraries were prepared for sequencing using standard Illumina protocols.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiSeq 2500

Data processing

Filtering and trimming (in order shown): removal of first 11 bases, clipping of remaining adapter sequences and removal of reads <25 bases, all done using Trimmomatic.
Reads were mapped to the P. acanthamoebae UV-7 genome using BWA (default settings).
Strand-specific reads per predicted gene were counted via HTSeq (union mode).
Normalised gene expression values and differential gene expression were determined using DESeq2.
Genome_build: Parachlamydia acanthamoebae UV-7 (Accession number: NC_015702).
Supplementary_files_format_and_content: UV7_raw_counts: tab-delimited text file includes raw counts data by gene (rows) and sample (columns).
Supplementary_files_format_and_content: UV7_log2(TPM): tab-delimited text file includes log2(TPM) values by gene (rows) and sample (columns).

Submission date

Sep 27, 2019

Last update date

Jan 20, 2020

Contact name

Matthias Horn

E-mail(s)

[email protected]

Organization name

University of Vienna

Department

Division of Microbial Ecology