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Sample GSM2093305

Query DataSets for GSM2093305

Status

Public on Sep 14, 2016

Title

ID-003036_bystanders

Sample type

SRA

Source name

Bone-marrow derived macrophages

Organism

Mus musculus

Characteristics

group: Bystanders
strain: C57/BL6
age: 8-12 week old

Treatment protocol

S. Typhimurium SL1344 strain harboring the pFCcGi plasmid (Figueira et al., 2013) was used for Fluorescence Dilution (FD) (Helaine et al., 2014). The strain was cultured for 16 h at 37°C in a minimum medium MgMES supplemented with 0.2% arabinose and 50 µg/mL ampicillin. Before infection, 180 µL of bacteria culture were opsonized 20 min at RT by supplementing 20 µL of mouse serum and 80 µL of differentiation medium.

Growth protocol

Bone marrow derived MΦs were obtained by isolation and differentiation of the marrow of the hind-leg bones of 8-12 week old C57/BL6 wild-type mice (Janvier labs). Briefly, a bone marrow suspension in X-Vivo 15 medium (Lonza), supplemented with 10 % fetal bovine serum (FBS superior, Biochrom) and 10 % L929 conditioned DMEM medium (herein called ‘differentiation medium’) was seeded into 6-well plates at a density of 106 leukocyte cells in 2 ml per well. Cells were incubated at 37 °C in a CO2-incubator with humidified atmosphere up to 7 days. At day 3 the cultures were supplemented with 0.3 volumes of fresh differentiation medium. At day 7, cells were harvested and seeded into 6 well tissue culture plates at a density of 106 cells per well in a fresh 2 mL differentiation medium.

Extracted molecule

total RNA

Extraction protocol

Single-cell libraries were prepared as reported in Smartseq2 protocol (Picelli et al., 2013; Picelli et al., 2014) with some technical adaptation: 1µL of ERCC spike-Mix 1 (Life Technologies) diluted at 1:106 in RNAse-free water was added after cell sorting to the lysis buffer; template-switching oligo (TSO) was modified to add isomeric nucleotide at the 5’end to minimize background cDNA synthesis as previously described (Kapteyn et al., 2010) (5’-(iso-dC)(iso-dG)(iso-dC)AAGCAGTGGTATCAACGCAGAGTACATrGrG+G-3’, Eurogentec); after first strand synthesis primer-dimers were removed using 11 µL of Ampure XP beads (Beckman Coulter). After the cDNA amplification, libraries were quantified using Qubit Hs Assay (Life Technologies) and quality of libraries were checked using Bioanalyser (Agilent)
Using the modified protocol described in (Patel et al., 2014), 1 ng of cDNA was subjected to tagmentation-based protocol (Nextera XT, Illumina) using ¼ of the recommended volumes, 10 min for tagmentation at 55°C and 1 min extension time during PCR. After PCR and cDNA purification, cDNA was purified and resuspended in 15 µL of elution buffer (EB) (Qiagen). Finally, libraries were pooled (16 libraries for Miseq sequencing and sequencing was performed in paired-end mode for 2 x 75 cycles using Illumina's MiSeq.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina MiSeq

Data processing

Illumina adapters and SMARTSeq2 PCR primer (5'-AAGCAGTGGTATCAACGCAGAGT-3') were removed using cutadapt (v1.3) (Martin et al. 2011).
Reads below a phred score of 20 were trimmed using the program fastq_quality_trimmer from the FASTX toolkit (v 0.0.13).
After quality trimming and adapter clipping unmatched paired end reads were removed using inhouse scripts.
Trimmed reads were mapped to the mouse genome reference sequence (GRCm38.p3) and 92 ERCC spike-ins sequences.
Alignment was performed using Bowtie2 (v 2.2.4) (Langmead and Salzberg, 2012) and Tophat (v 2.0.13) (Trapnell et al., 2009) with default settings. The incorrectly annotated transcript ENSMUSG0000092329 was removed from the mouse annotation file.
Read counts of each gene were determined using the HTSeq program (Anders et al., 2015).
Genome_build: GRCm38.p3
Supplementary_files_format_and_content: text file containing raw read counts

Submission date

Mar 18, 2016

Last update date

Apr 12, 2023

Contact name

Silke Appenzeller

E-mail(s)

silke.appenzeller@uni-wuerzburg.de

Organization name

University Wuerzburg

Street address

Schweinfurter Str. 28