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Sample GSM5266038

Query DataSets for GSM5266038

Status

Public on Aug 23, 2021

Title

3015_LPS_IFNg_wt

Sample type

SRA

Source name

microglia

Organism

Mus musculus

Characteristics

strain: C57BL/6J
tissue: brain
developmental stage: neonatal
genotype: WT
agent: LPS_IFNg

Treatment protocol

Briefly, after decapitation, extracted brains were collected in ice-cold PBS. Cortices of both hemispheres were collected after removal of meninges and triturated until a single-cell suspension was achieved. Isolated cells were cultivated as mixed glia culture until they reached confluency after approximately 14 days. Cells were cultivated in DMEM high glucose (Gibco, Darmstadt, Germany) containing 10 % fetal calf serum (FCS) (PAA, Freiburg, Germany), 1 % penicillin/streptomycin (Gibco). The medium was changed twice weekly. Microglia cells were harvested by shaking for 1 h at 200 rpm and re-seeded in 24-well plates for stimulation experiments at a density of 1.5 x 105 cells/ml. After re-seeding, cells were rested for 24 h.
For stimulation experiments, cells were treated for 30 min or 16 h with E. coli LPS serotype 0127:B8 (100 ng/ml) (Sigma-Aldrich, Taufkirchen, Germany) and IFNγ (20 ng/ml) (R&D Systems, Wiesbaden, Germany), CpG (1 nmol/mL), or PolyI:C (50g/mL).

Extracted molecule

total RNA

Extraction protocol

miRNeasy micro Kit (Qiagen) according to the manufacturers' recommendations. Total RNA was eluted in RNAse free water.
Samples with more than 100 ng total RNA were converted into NGS libraries using the TruSeq RNA Library Prep Kit v2 (Illumina) at 100 ng input according to manufacturer’s recommendations. The size-distribution of the libraries was determined using the Agilent D1000 assay on a Tapestation 2200 system (Agilent). Libraries were quantified using a Qubit HS dsDNA assay. 75 bp single-end sequencing was performed on a HiSeq1500 system using Rapid v2 chemistry. Base calling from base call files and demultiplexing was performed with CASAVA v1.8 (Illumina). Low input Samples were converted into libraries of double stranded cDNA molecules as a template for high throughput sequencing following the SMART-Seq2 protocol. Shortly, mRNA was primed for SMART reverse transcription from 5 ng of total RNA using poly-T oligos. cDNA was pre-amplified by SMART ISPCR. Fragmentation was performed using the Illumina Nextera XT kit, followed by PCR amplification and indexing. Size-selection and purification of library fragments preferentially 300-400 bp in length was performed using SPRIBeads (Beckman-Coulter). The size-distribution of cDNA libraries was measured using the Agilent high sensitivity D5000 assay on a Tapestation 4200 system (Agilent). cDNA libraries were quantified using a Qubit high sensitivity dsDNA assay. 75 bp single-end sequencing was performed on a NextSeq500 system using High Output v2.5 chemistry. Base calling from base call files and file conversion to fastq files were achieved by Illumina standard pipeline scripts (bcl2fastq2 v.2.20).

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiSeq 1500

Description

3015_LPS_IFNg_wt
3015

Data processing

base calling and de-multipexing using CASAVA version 1.8
Alignment and quatification with Kallisto l using the default parameters
reads mapped to each transcript against the RefSeq mm38 annotation download on March 2018
Normalization using DeSeq2
Genome_build: mm38
Supplementary_files_format_and_content: comma separated files include DeSeq2 normalized counts for each Sample.
Supplementary_files_format_and_content: comma separated files log ttransformed counts corrected for the experimental day for each Sample.

Submission date

Apr 26, 2021

Last update date

Aug 23, 2021

Contact name

Joachim Schultze

E-mail(s)

j.schultze@uni-bonn.de

Organization name

LIMES (Life and Medical Sciences Center Genomics and Immunoregulation)

Department

Genomics and Immunoregulation