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Status |
Public on Jun 21, 2018 |
Title |
LPS_IC_DAPT_REP1_003 |
Sample type |
SRA |
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Source name |
Bone marrow
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Organism |
Mus musculus |
Characteristics |
strain: C57BL/6 tissue: Bone marrow age: 8-10 weeks
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Treatment protocol |
BMDMs were primed overnight with recombinant murine IFNg (10 ng/mL) (BioLegend, USA) and washed twice by media and warm PBS, respectively. Salmonella LPS (100 ng/mL) (Sigma-Aldrich, USA), rabbit IgG against ovalbumin (OVA) (GeneTex, USA) and IgG-opsonized OVA (immune complex) were added to activate macrophages as indicated. Gamma secretase inhibitor (GSI), DAPT (25 µM) (Calbiochem, USA) and vehicle control DMSO (0.01%) (Sigma-Aldrich, USA) were added to macrophages.
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Growth protocol |
To generate bone marrow derived macrophages (BMDMs),bone marrow cells were plated in non-tissue culture treated plates (Hycon, Thailand) and cultured at 37°C, 5% CO2 in 8 mL of media containing DMEM supplemented with 10% Fetal Bovine Serum, HEPES, sodium pyruvate , streptomycin/penicillin G (all from LONZA, USA or Hyclone, UK), 5% (v/v) horse serum (Thermo Scientific, USA) and 20% (v/v) L929-conditioned media. Three mL of fresh DMEM media supplemented with 20% L929-conditioned media and 5% horse serum was added to the culture at day 4. Cells were harvested at the day 7 and used for all the experiments.
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Extracted molecule |
total RNA |
Extraction protocol |
Total RNA samples were extracted from LPS/IC-activated BMDMs in the presence of DMSO or GSI for 1 hr using an RNeasy Mini Kit (Qiagen, USA). RNA samples were assessed for quality using an Agilent 2100 Bioanalyzer (Agilent, USA) and quantified using a Qubit 3.0 fluorometer (Life Technologies, USA). RNA libraries were prepared for sequencing using standard Illumina protocols
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Library strategy |
RNA-Seq |
Library source |
transcriptomic |
Library selection |
cDNA |
Instrument model |
Illumina NextSeq 500 |
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Data processing |
bcl2fastq2 Conversion Software v2.20 used for basecalling. Sequenced reads were trimmed for adaptor sequence, and then mapped to mm9 whole genome using STAR version 2.5.0a with parameters --runMode alignReads --runThreadN 12 --readFilesCommand zcat --outStd Log --outSAMunmapped within --outSAMtype BM SortedByCordinate limitGenomeGenerateRAM 40,000,000 --limitIObufferSize 40,000,000 --outWigType wiggle --chimSegmentMin 21 bam files were merge using picard and remove duplicate or PCR product contamination Raw count from each samples were generated using HT-Seq program with parameters --HTSeq.GenomicArrayOfSets("auto", stranded = True) Genome_build: mm10 Supplementary_files_format_and_content: tab-limited files with raw counts value
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Submission date |
May 03, 2018 |
Last update date |
Jun 21, 2018 |
Contact name |
Tanapat Palaga |
E-mail(s) |
tanapat.p@chula.ac.th
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Phone |
6622185070
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Organization name |
Chulalongkorn University
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Department |
Microbiology
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Lab |
2015
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Street address |
Payathai Road, Pathumwan
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City |
Bangkok |
ZIP/Postal code |
10330 |
Country |
Thailand |
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Platform ID |
GPL19057 |
Series (1) |
GSE114020 |
Effect of Notch Signaling Inhibitor on Gene Expression in Lipopolysaccharide-stimulated Macrophages in the Presence of Immune Complex |
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Relations |
BioSample |
SAMN09060752 |
SRA |
SRX4037338 |
Supplementary data files not provided |
SRA Run Selector |
Raw data are available in SRA |
Processed data are available on Series record |
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