|
| Status |
Public on Feb 16, 2022 |
| Title |
NFD 2 |
| Sample type |
SRA |
| |
|
| Source name |
Tumor associated macrophages
|
| Organism |
Mus musculus |
| Characteristics |
tissue: Prostate tissue
condition: Normal diet
|
| Treatment protocol |
Mice were injected subcutaneously with 2.5x10^6 prostatic cancer cells (Pten -/-; Smad4 -/-). After tumor onset (10 days), mice randomly divided into two groups: NFD and HFD. After 45 days mice were sacrified.
|
| Growth protocol |
not applicable
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Tumoral tissue was dissociated to single cell suspension. Briefly, tumors were digested for 1 hour and half in a Collagenase type V solution (1 mg/ml) at 37°C, 5% CO2 on a rocking platform. Cells digestion was then pelleted and incubated in 2.5% Trypsin for five minutes at 37°C, 5%CO2. Trypsin was then inactivated by complete medium with FBS and cells suspension was further passed through a syringe needle until complete dissociation and filtered through a 40 μm cell strainer. CD45+CD11b+F480+Ly6G- cells were sorted by flow cytometry, resuspended in 1ml PBS plus 0.04% BSA and washed two times by centrifugation at 450 rcf for 7min. After the second wash cells were resuspended in 250 ul of Trizol and RNA was extracted according to standard procedures
Total-RNA-sequencing library preparation was performed starting from 0.5 ng of total-RNA with the SMART-Seq Stranded Kit (Clontech-Takara). Libraries were obtained and qualitatively assessed by using TapeStation 4200 and quantified by Qubit Fluorimeter. Afterwards, they were multiplexed in an equimolar pool and sequenced on a NextSeq-500 Illumina Platform generating at least 40 million 75bp-PE reads per sample (i.e. 80 million reads per sample).
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
NextSeq 550 |
| |
|
| Data processing |
Bcl2fastq (v2.19.0.316) for demultiplexing and conversion to Fastq files
Raw reads were preprocessed for adapter trimming and quality check was assessed using the FastQC tool (http://www.bioinformatics.babraham.ac.uk/projects/fastqc). Reads were aligned to the reference genome (Ensembl Mus Musculus release mm10) using the STAR algorithm (version 020201) (Dobin et al., 2013). The featureCounts package (version 1.6.4) implemented in the Rsubread package was used to estimate read counts along genes. Differential expression analysis was performed using the GLM approach implemented in the R/Bionconductor edgeR (Robinson et al., 2010) package (R version 3.5; edgeR version 3.24.3).
Genome_build: mm10
Supplementary_files_format_and_content: bigWig files
|
| |
|
| Submission date |
Jul 07, 2020 |
| Last update date |
Feb 16, 2022 |
| Contact name |
Roberta Carriero |
| E-mail(s) |
roberta.carriero@humanitasresearch.it
|
| Organization name |
Humanitas Research Hospital
|
| Street address |
Via Rita Levi Montalcini,4
|
| City |
Pieve Emanuele |
| State/province |
MI |
| ZIP/Postal code |
20090 |
| Country |
Italy |
| |
|
| Platform ID |
GPL21626 |
| Series (1) |
| GSE153977 |
Transcriptional profile of tumor associated macrophages in High fat diet mouse models |
|
| Relations |
| BioSample |
SAMN15468461 |
| SRA |
SRX8683547 |
