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Sample GSM6153671

Query DataSets for GSM6153671

Status

Public on May 19, 2022

Title

24h_Vneg_M5 [21]

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-

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