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Sample GSM2464545

Query DataSets for GSM2464545

Status

Public on Apr 26, 2017

Title

96 hpi, replicate 2

Sample type

SRA

Source name

amoeba cells infected with endosymbionts

Organisms

Acanthamoeba castellanii; Candidatus Protochlamydia amoebophila

Characteristics

a. castellanii strain: Neff
p. amoebophila strain: UWE25
developmental stage of endosymbiont: 96 hours post infection
multiplicity of infection: 15

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, amoebae were 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 time points. Uninfected amoebae were harvested with the 2 hpi samples.

Growth protocol

Amoebae with and without the endosymbiont were maintained at 20°C in PYG (20 g/l proteose peptone, 100 mM glucose, 2 g/l yeast extract, 1 g/l sodium citrate dihydrate, 4 mM MgSO4*7H2O, 1.32 mM Na2HPO4*2H2O, 2.5 mM KH2PO4, 0.05 mM Fe(NH4)2(SO4)2*6H2O; pH 6.5). For infection, uninfected amoebae were seeded at low density 3 days prior to infection and were then allowed to grow as above, before purified, extracellular endosymbionts were added. Two hours after infection cultures were washed 3x and infected cultures were harvested or further incubated at 20°C in PYG for 48 and 96 hours. Purified, extracellular endosymbionts were obtained from amoeba-endosymbiont cultures that were grown at the same conditions, and extracellular endosymbionts were harvested from the medium supernatant after one week of incubation.

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).
Ribosomal RNA was removed using the RiboZero Magnetic kit (Illumina) for Gram-positive bacteria as recommended by the manufacturer. To enrich for mRNA from symbiont-free amoebae, the RNA was additionally treated with Dynabeads mRNA Purification Kit (Thermo Fisher Scientific). For strand-specific cDNA library preparation, the NEBNext Ultra Directional RNA Library Prep Kit for Illumina in combination with the NEBNext Multiplex Oligos (New England Biolabs) was used.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiSeq 2000

Description

UWE25_log2rpkm.txt
Neff_log2rpkm.txt

Data processing

Filtering and trimming (in order shown): removal of first 13 bases (PRINSEQ-lite), quality trimming (mothur), removal of reads containing >2 ambiguous bases (PRINSEQ-lite), removal of poly-A tails (PRINSEQ-lite), clipping of remaining adapter sequences (Biopieces), removal of reads <25 bases;
Reads were mapped to the P. amoebophila genome using BWA (default settings). For the mapping to the A. castellanii genome TopHat was used (default settings, with the options fr-firststrand and -G gff.file).
Strand-specific reads per predicted gene were counted via HTSeq (union mode).
Normalized gene expression values and differential gene expression were determined using edgeR.
Genome_build: Protochlamydia amoebophila UWE25 (Accession number: BX908798)
Genome_build: Acanthamoeba castellanii Neff (Accession number: AHJI00000000)
Supplementary_files_format_and_content: log2RPKM values by gene (rows) and time point (columns)

Submission date

Jan 20, 2017

Last update date

May 15, 2019

Contact name

Lena König

E-mail(s)

[email protected]

Organization name

University of Vienna

Street address

Althanstrasse 14