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Sample GSM4670341

Query DataSets for GSM4670341

Status

Public on Apr 28, 2021

Title

9_S25: Uninfected 24h replicate 2

Sample type

SRA

Source name

Chicken macrophage cell culture

Organism

Gallus gallus

Characteristics

infection status: Uninfected
sample time post-infection [hours]: 24
chicken cell line: Macrophage HD-11
e. tenella strain: --

Treatment protocol

At infection, 4.5 x106 sporozoites per well in 6-well plates and 0.28 x106 sporozoites per well in 96-well plates were added to the cultures aiming at a ratio of 4 sporozoites per HD11 cell. The same volume of growth medium without parasites was added to uninfected control cultures. Plates were then cultured until RNA harvest up to 72 h and for plates cultured longer than 24 h, growth medium was removed at 24 h, cultures washed with phosphate buffered saline (PBS; without Ca2+ and Mg2+ at pH 7) to remove loose sporozoites and fresh growth medium was added to the wells before culture was continued.

Growth protocol

The immortalised chicken macrophage cell-line HD11 (Beug et al., 1979) was maintained in growth medium, i.e. RPMI 1640 (National Veterinary Institute) supplemented with 200 IU penicillin/ml, 100 µg streptomycin/ml, 2mM L glutamine, and 5% FCS. Before infection HD11 cells were trypsinised and live cells were counted by trypan blue exclusion. Cells were seeded at 5.6 x105 cells per well in flat-bottomed 6-well tissue culture plates (Nunc™, ThermoFisher Scientific) in 2 ml of growth medium and at 0.35 x105 cells per well in flat-bottomed 96-well tissue culture plates (Nunc™, ThermoFisher Scientific) in 100 µl of growth medium. Plates were incubated at 40°C, 5.2% CO2 in air at a humid atmosphere for 24 h after which the HD11 cells were approx. 50-70 % confluent in the wells. Cryopreserved E. tenella sporozoites were thawed into RPMI 1640 medium with 15% FCS, centrifuged down at 910 x g for 7 min, resuspended in growth medium and viable sporozoites were counted by trypan blue exclusion

Extracted molecule

total RNA

Extraction protocol

For RNA isolation 1 ml of HD11 cell lysate in TRIzol was used and RNA was extracted using chloroform according to the TRIzol manufacturer’s protocol. The Isolated RNA was subsequently treated with DNAse (TURBO™ DNase, 2U/ul, ThermoFisher Scientific) according to the manufacturers protocol and further purified using reagents and the “RNA cleanup” protocol of the RNeasy Mini kit (Qiagen) protocol. RNA concentration and quality was then assessed using the Agilent RNA 6000 Nano kit on a 2100 Bioanalyzer Instrument (Agilent) and the RNA stored at -70°C until further analysis.
Sequencing libraries were prepared from 0.12 – 0.5 μg total RNA using the TruSeq stranded mRNA library preparation kit (Cat# RS-122-2101/2102, Illumina Inc.) including polyA selection. The library preparation was performed according to the manufacturers’ protocol (#15031047). Sequencing: HiSeq2500, paired-end 125bp read length, v4 sequencing chemistry.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiSeq 2500

Data processing

Base calling with Illumina basecaller
Reads were trimmed to get rid of adapters and low-quality parts with Trimmomatic v. 0.36, using the standard Illumina adapter library, SLIDINGWINDOW:4:20 and MINLEN:50
Reads were mapped to a merged version of the reference genomes (effectively mapping to both at the same time as if they were a single genome) with STAR v. 2.7.2b, using default settings
Reads mapping to features were counted using HTSeq v. 0.9.1, with strandedness set on reverse, i.e. -s reverse, and otherwise default settings
Read counts loaded into R and, using edgeR, each sample was CPM normalized with regards to library size and genes filtered out that had few/no reads mapping to them across samples
Genome_build: GRCg6a/Eth001
Supplementary_files_format_and_content: comma separated text files containing raw gene counts for each gene and sample, one for the chicken data and one for E. tenella
Supplementary_files_format_and_content: comma separated text files containing CPM normalized read counts for each organism, with low expressed genes across time points filtered out along with samples without chicken data, for the chicken dataset, and samples without E. tenella data, for the E. tenella dataset

Submission date

Jul 14, 2020

Last update date

Apr 28, 2021

Contact name

Arnar Kari Sigurdarson Sandholt

E-mail(s)

a.k.sigurdarsonsandholt@uu.nl

Organization name

Utrecht University

Department

IRAS