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| Status |
Public on Feb 18, 2024 |
| Title |
Neutrophils PBS rep1 |
| Sample type |
SRA |
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| Source name |
bone marrow-derived
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| Organism |
Mus musculus |
| Characteristics |
tissue: bone marrow-derived
cell type: neutrophils
treatment: PBS (Saline control)
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| Treatment protocol |
Freshly isolated neutrophils were treated with CGRP (1 nM) in RPMI with 10% FBS and 100 units/ml penicillin/streptomycin for 4 hours at 37°C with 5% CO2. Freshly isolated monocytes were cultured in DMEM/F12 with 10% FBS, 100 units/ml penicillin/streptomycin, and M-CSF (10 ng/ml) for 3 days. The cell culture medium was replaced with medium containing CGRP (1 nM) for 4 hours at 37°C with 5% CO2.
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| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was extracted using the RNeasy Plus Micro Kit (Qiagen, #74034) according to the manufacturer’s instructions
RNA samples (20 ng) with RIN value ≥ 7 were used for library preparation. First strand synthesis was performed using a dT primer which also adds the Illumina P7 (5ʹ CAA GCA GAA GAC GGC ATA CGA GAT 3ʹ), 8 bp i7 index for each sample and a 10 bp unique molecular identifier (UMI). The modified reverse transcriptase reaction also adds a template switching sequence at the 5’ end of the RNA during the generation of indexed cDNA. These first stand indexed cDNA were pooled and amplified using primers to P7 and the template switch sequence. The Illumina P5 was added by tagmentation by Nextera transposase during amplification. The standard Illumina R1 primer was used (main cDNA read), followed by standard i7 primer for index/UMI. The R2 primer was present but not used as it will read into poly-A tail.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
NextSeq 2000 |
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| Description |
Processed_Neutrophil_data.csv
Neu_PBS1
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| Data processing |
Fastq files were processed using the nfCore/RNAseq (v3.2) pipeline using the umi function (Patel, H., Ewels, P. & Peltzer, 2013, A. nf-core/rnaseq: nf-core/rnaseq v3.2 - Copper Flamingo, Zenodo)
Reads were aligned to the Mus musculus GRCm38 reference using STAR aligner (Dobin, A., et al., 2013, Bioinformatics)
Reads were quantified using featureCounts (Liao, Y., et al., 2014, Bioinformatics) producing the raw genes count matrix and various quality control metrics.
Raw counts were then analysed with Degust web tool (Powell, D.R., et al., 2019, Zenodo) which performs normalisation using trimmed mean of M values (TMM) (Robinson, M.D. and Oshlack, A., 2010, Genome Biol) and differential gene expression analysis using limma/voom (Law, C.W., et al., 2014, Genome Biol)
Assembly: GRCm38
Supplementary files format and content: csv file where rows represent genes and each column represents different information, including gene name, gene ID and raw gene counts corresponding to the individual samples.
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| Submission date |
Feb 05, 2024 |
| Last update date |
Feb 18, 2024 |
| Contact name |
Mikaël Martino |
| E-mail(s) |
mikael.martino@monash.edu
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| Organization name |
Monash University
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| Department |
Australian Regenerative Medicine Institute
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| Lab |
Martino Lab
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| Street address |
15 Innovation Walk, Clayton, VIC
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| City |
Clayton |
| State/province |
VICTORIA |
| ZIP/Postal code |
3800 |
| Country |
Australia |
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| Platform ID |
GPL30172 |
| Series (1) |
| GSE255049 |
CGRP sensory neurons promote tissue healing by modulating neutrophils and macrophages |
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| Relations |
| BioSample |
SAMN39829920 |
| SRA |
SRX23533637 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
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