GEO Accession viewer

NCBI > GEO > Accession Display

Not logged in | Login

GEO help: Mouse over screen elements for information.

Sample GSM5772481

Query DataSets for GSM5772481

Status

Public on May 25, 2022

Title

Macrophage-MeganVac1-R1

Sample type

SRA

Source name

Primary macrophage cells

Organism

Gallus gallus

Characteristics

cell type: PBMC derived macrophages
infection: Infected with UK1-cya crp

Treatment protocol

Transcriptome analyses were performed using seven-day old, macrophages differentiated as described above (Huang et al., 2019). Approximately 5 × 104 macrophages were seeded in a 24-well plate and incubated overnight at 41°C with 5% CO2. Overnight grown S. Typhimurium cells were concentrated and washed twice with DPBS. Approximately 1 × 106 bacteria were then resuspended in 1 mL of 10% chicken serum in DPBS and incubated for 30 minutes at 37°C. Bacterial cells were then washed twice in DPBS and resuspended in 1 mL RPMI 1640 medium with 10% heat-inactivated FBS. The primary macrophages were infected with the S. Typhimurium mutants with an MOI of 10 for 30 minutes. After 30 minutes, extracellular bacterial were killed by adding 100 µg/mL of gentamicin for an hour and the medium was then replaced with fresh RPMI-1640 medium supplemented with 30 µg/mL gentamicin and incubated for another hour. Cells were then washed twice with DPBS, lysed by using the RLT buffer (Qiagen) and shipped on dry ice to Cofactor Genomics for RNA-seq

Growth protocol

Chicken PBMCs were isolated and cultured according to a previous protocol (Feng et al., 2017, Wigley et al., 2002, Peng et al., 2020). Briefly, blood was collected from healthy chickens and heparinized blood was mixed with an equal volume of DPBS. Ten mL of this suspension was then carefully layered on top of 10 mL Histopaque®-1077 (Sigma) in a 50 mL conical tube. Tubes were then centrifuged at 400 × g for 30 minutes at room temperature with the lowest acceleration and no braking. After centrifugation, the top plasma layer was removed and the interface was collected in a fresh tube. The collected lymphocytes were then mixed gently with 5 volumes of DPBS and centrifuged at 250 × g for 10 minutes at room temperature. The supernatant was discarded, and the pellet was washed twice with DPBS at room temperature. The pellet was then finally resuspended in RPMI 1640 Medium, supplemented with GlutaMAX™ (ThermoFisher Sc.), 10% FBS (Gibco), Anti-anti (ThermoFisher Sc.) and 50 µg/mL chicken granulocyte-macrophage colony-stimulating factor (GMCSF, Abcam). The viability of the cells was determined by Trypan Blue exclusion and approximately 1 × 107 cells were transferred to a 24 well plate and incubated at 41°C with 5% CO2. The spent medium was replaced with a fresh medium supplemented with GMCSF every 2 days and the morphology of the adherent cells was monitored using an inverted microscope. After 7 days of incubation, the cells were dislodged from the surface and analyzed using flow cytometry. Macrophages were identified by staining the cells with chicken macrophage marker KUL01 (ThermoFisher Scientific) and analyzing using a flow cytometer (BD FACSAria). as described previously (Mast et al., 1998).

Extracted molecule

total RNA

Extraction protocol

The samples were processed by Cofactor Genomics as described previously (do Amaral et al., 2020). Briefly, total RNA was isolated using Qiagen RNAeasy Mini Kit (Qiagen) following the manufacturer’s instructions. The isolated RNA was then treated with DNase I (Thermo Fisher Scientific) to remove potential DNA contamination. RNA was then precipitated using lithium chloride (Sigma). The concentration and quality of RNA were determined using nanophotometer.
RNA samples with RNA integrity number (RIN) > 8.0, A260/A280 > 1.9 and A260/A230 > 2 were selected for mRNA library preparation and Next Generation Sequencing. Briefly, the total RNA was incubated with mRNA capture beads to remove contaminating ribosomal RNA from the sample using the Kapa Stranded mRNA-Seq kit (Kapa Biosystems) following manufacturer’s instructions. The resulting poly(A)-captured mRNA was then fragmented. First-strand cDNA synthesis was performed using reverse transcriptase and random primers in the presence of Actinomycin D, followed by second-strand cDNA synthesis with DNA polymerase I and RNase H. Double-stranded cDNA was end-repaired and A-tailed for subsequent adaptor ligation. Indexed adaptors were ligated to the A-tailed cDNA. Enrichment by PCR was performed to generate the final cDNA sequencing library. Libraries were sequenced as paired-end 150 base reads on an Illumina NextSeq 500 following the manufacturer's protocols.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina NextSeq 500

Data processing

Illumina Casava1.7 software used for basecalling.
Reads were aligned, quantified and analyzed using CLC Genomics Workbench (Qiagen).
Differential gene expression among samples was determined using the DESeq2 package for R in Rstudio (v. 1.0.136). Data were normalized with DESeq2 algorithms.
Transcripts with less than one raw count were excluded, and the default DESeq2 algorithms were used to remove outlier transcripts based on Cook’s distances. The Benjamini and Hochberg algorithm was used to control the false discovery rate (FDR) and transcripts were considered differentially expressed if the expression between two treatments differed by at least 2-fold with FDR < 0.1 (Li et al., 2012).
Genome_build: GRCg6a (GCA_000002315.5)
Supplementary_files_format_and_content: Tab delimited text files of pairwise transcriptome including FDR valus, fold change.

Submission date

Jan 05, 2022

Last update date

May 25, 2022

Contact name

Bijit Kumar Bhowmik

E-mail(s)

bijitkrbhowmik@gmail.com

Organization name

Elanco Animal Health

Department

Discovery Bacteriology & Microbiome