GEO Accession viewer

NCBI > GEO > Accession Display

Not logged in | Login

GEO help: Mouse over screen elements for information.

Sample GSM3583846

Query DataSets for GSM3583846

Status

Public on Apr 14, 2019

Title

Neff/Pam+Lpn_96hpi_1

Sample type

SRA

Source name

amoebae cells containing the endosymbiont and infected with L. pneumophila

Organisms

Acanthamoeba castellanii; Candidatus Protochlamydia amoebophila

Characteristics

a. castellanii strain: Neff
p. amoebophila strain: UWE25
l. pneumophila strain: Paris
condition: L. pneumophila
time point: 96 hours post infection
l. pneumophila multiplicity of infection (moi): 3

Treatment protocol

Cultures were infected by adding L. pneumophila directly to the flasks. Symbiont-free and symbiont-containing cultures, as well as uninfected symbiont-containing cultures were then incubated for 2 hours before amoebae were washed four times with infection buffer (Moffat & Tompkins, 1992). PYG medium was added, cultures were sampled at 2 hours post infection (hpi) to monitor initial infection efficiency by FISH, and were further incubated at 20°C for 24 and 96 h, respectively. Released, extracellular L. pneumophila were collected at 96 hpi by centrifuging the supernatant of infected cultures at 150 x g for 2 min to roughly separate amoebae from bacterial cells, then filtering the supernatant through 5 µm syringe filters (Sartorius) to remove residual amoebae, and finally pelleting the bacterial cells at 12,850 x g for 2 min. L. pneumophila infected amoebae as well as uninfected amoebae were harvested and bacteria were roughly enriched as previously described (König et al., 2016), but to optimize yield of intracellular bacteria, cell suspensions were additionally vortexed for 1 min together with smaller glass beads (diameter 0.25-0.5 mm, Carl Roth) after vortexing with larger beads (diameter 0.75 - 1 mm, Carl Roth).

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). Before infection with L. pneumophila, amoebae with and without endosymbionts were seeded (9x10^6 cells per flask) and allowed to grow for 3 days at 20°C. Infectious L. pneumophila were prepared as follows: ACES-buffered yeast extract (AYE) medium supplemented with 0.4 g/l L-cysteine and 0.135 g/l ferric nitrate was inoculated with L. pneumophila Paris, grown overnight at 37°C on a roller drum (Eppendorf, Hamburg, Germany), diluted with fresh medium to OD600 = 0.2-0.3, and grown to post-exponential phase (OD600 ≥ 3.5). Only cultures exhibiting a high proportion of motile cells were used for infection. Motility was assessed qualitatively as described previously (Byrne & Swanson, 1998). The number of viable L. pneumophila added to amoebae was determined by diluting the cultures in infection buffer (Moffat & Tompkins, 1992) and subsequent plating of dilutions on ACES-buffered charcoal yeast extract (CYE) plates in triplicate.

Extracted molecule

total RNA

Extraction protocol

Total RNA from extracellular bacteria (with and without symbionts) and intracellular bacteria enriched from co-cultures at 24 and 96 hpi (with and without symbionts) was extracted using TRIzol Reagent (Thermo Fisher Scientific), and residual DNA was digested using the Turbo DNA-free Kit (Thermo Fisher Scientific), both as described before (König et al., 2016). rRNA depletion using the Ribo-Zero Gold rRNA Removal Kit (Illumina) was performed by the Vienna Biocenter Core Facilities (VBCF) Next-Generation Sequencing (NGS) Unit (http://www.vbcf.ac.at).
Library preparation using the NEBNext Ultra RNA Library Prep Kit for Illumina (New England Biolabs) as well as sequencing on an Illumina HiSeq 2500 with 100 bp read length was performed by the Vienna Biocenter Core Facilities (VBCF) Next-Generation Sequencing (NGS) Unit (http://www.vbcf.ac.at).

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiSeq 2500

Description

Acanthamoeba castellanii/Protochlamydia amoebophila/Legionella pneumophila
Legionella_pneumophila_Paris.txt
Protochlamydia_amoebophila_UWE25.txt

Data processing

PRINSEQ-lite was used trimming and quality filtering of the raw reads: first 13 based were removed (-trim_left 13), reads were trimmed when the base calling quality decreased below a phred score of 20 within a window of 5 bases (-trim_qual_right 20, -trim_qual_window 5), reads containing more than two ambiguous bases (Ns) were removed (-ns_max_n 2), poly-A/T tails were trimmed (-trim_tail_left 5, -trim_tail_right 5), and all reads shorter than 25 bases were removed (-min_len 25).
Trimmed reads were mapped to the P. amoebophila and L. pneumophila genomes, respectively, using BWA (default settings). Using samtools, only uniquely mapped reads were kept (-q 10, -F 4).
Strand-specific reads per predicted gene were counted via HTSeq (union mode).
Differential gene expression (logFC >1, FDR < 0.05) was determined using edgeR.
Genome_build: NC_005861.1
Genome_build: NC_002942.5
Supplementary_files_format_and_content: raw read counts as output by HTSeq for each gene locus tag (rows) and sample (columns); one file for each organism

Submission date

Jan 30, 2019

Last update date

Apr 14, 2019

Contact name

Lena König

E-mail(s)

lena.koenig@univie.ac.at

Organization name

University of Vienna

Street address

Althanstrasse 14