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Sample GSM6153670 Query DataSets for GSM6153670
Status Public on May 19, 2022
Title 24h_Vneg_IFNb_M3 [15]
Sample type SRA
 
Source name Bone marrow derived macrophages
Organism Mus musculus
Characteristics cell type: Bone marrow derived macrophages
strain: C57BL/6
genotype: Wild type
time point: 24h
treatment: LgyLRV1- and IFNb
Treatment protocol One day after their plating, murine BMDMs were infected with stationary-phase Leishmania promastigotes with a multiplicity of infection (MOI) of 3. Co-treatment with LCMV was done with MOI of 1. Cells were also alternatively treated with IFNa or IFNb. The cells were incubated at 35°C and 5% CO2.
Growth protocol Macrophages were isolated from bone marrow of C57BL/6 mice (wild-type). Hind leg bones were collected and the bone marrow was flushed out with complete DMEM medium, containing DMEM (Dulbecco’s modified Eagle’s medium, Gibco), supplemented with 10% heat-inactivated FBS (Gibco), 1% Penicillin-Streptomycin Solution (BioConcept) (i.e. 100 IU/ml and 100 µg/ml respectively), 1% HEPES (BioConcept) (i.e. 10mM). The cells were differentiated into macrophages for 6 days in complete DMEM medium containing 50 ng/ml recombinant mouse Macrophage Colony Stimulating Factor (rmM-CSF, ImmunoTools), at 37°C and 5% CO2, in Sterilin Petri Dish (Thermo Scientific). Then, the bone marrow-derived macrophages (BMDMs) were detached with 1X DPBS (Gibco) containing 5 mM EDTA, washed and resuspended with complete DMEM medium. The cells were plated in 12-well plates (TPP) with 1.95 million cells per well. They were finally put back in culture at 37°C and 5% CO2.
Extracted molecule total RNA
Extraction protocol BMDMs were cleared out of supernatants and lysed with 350 µl RLT buffer (Qiagen) supplemented with 40 mM DTT (Dithiotreitol) for RNA extraction. Plates were then frozen at -80°C until the RNA purification. The RNA samples were purified with the RNeasy Plus Mini Kit (Qiagen) following the manufacturer’s instructions. Purified RNA was eluted with 30 µl RNase-free water (Qiagen).
Library preparation and sequencing were performed at the Lausanne Genomic Technologies Facility (GTF), University of Lausanne (UNIL), Switzerland
 
Library strategy RNA-Seq
Library source transcriptomic
Library selection cDNA
Instrument model Illumina HiSeq 2500
 
Data processing Purity-filtered reads were adapters and quality trimmed with Cutadapt (v. 1.8, Martin 2011).
Reads matching to ribosomal RNA sequences were removed with fastq_screen (v. 0.11.1). Remaining reads were further filtered for low complexity with reaper (v. 15-065, Davis et al. 2013)
Reads were aligned against the Mus musculus.GRCm38.92 genome using STAR (v. 2.5.3a, Dobin et al. 2013). The number of read counts per gene locus was summarized with htseq-count (v. 0.9.1, Anders et al. 2014) using Mus musculus.GRCm38.92 gene annotation.
Quality of the RNA-seq data alignment was assessed using RSeQC (v. 2.3.7, Wang et al. 2012). Reads were also aligned to the Mus musculus.GRCm38.92 transcriptome using STAR (v. 2.5.3a, Dobin et al. 2013) and the estimation of the isoforms abundance was computed using RSEM (v. 1.2.31, Li and Dewey 2011).
Statistical analysis was performed for genes independently in R (R version 3.4.4). Genes with low counts were filtered out according to the rule of 1 count(s) per million (cpm) in at least 1 sample. Library sizes were scaled using TMM normalization. Subsequently, the normalized counts were transformed to cpm values, and a log2 transformation was applied, by means of the function cpm with the parameter setting prior.counts = 1 (EdgeR v 3.20.9; Robinson et al. 2010).
Assembly: Mus musculus GRCm38.92 genome
Supplementary files format and content: tab-delimited text files (normalized logcpm)
 
Submission date May 16, 2022
Last update date May 19, 2022
Contact name Nicolas Fasel
E-mail(s) nicolas.fasel@unil.ch
Organization name University of Lausanne
Department Department of Biochemistry
Street address Chemin des Boveresses 155
City Epalinges
ZIP/Postal code 1066
Country Switzerland
 
Platform ID GPL17021
Series (1)
GSE203088 Macrophage response towards infection by the Leishmania-viral endosymbiont duo
Relations
BioSample SAMN28448329
SRA SRX15285291

Supplementary data files not provided
SRA Run SelectorHelp
Raw data are available in SRA
Processed data are available on Series record

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