|
| Status |
Public on Feb 16, 2022 |
| Title |
Ptenpc-/-;Smad4pc-/- 2 |
| Sample type |
SRA |
| |
|
| Source name |
Tumor associated macrophages
|
| Organism |
Mus musculus |
| Characteristics |
tissue: Prostate tissue
genotype: Ptenpc-/-;Smad4pc-/-
|
| Treatment protocol |
not applicable
|
| Growth protocol |
not applicable
|
| Extracted molecule |
total RNA |
| Extraction protocol |
murine prostates were 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 procedure.
RNA quality control was performed with the Agilent 2200 Tape Station system and only RNAs having a RIN>8 were used for library preparation. Libraries for mRNA sequencing were prepared starting from 5 ng tot RNA for each sample by using the SMART-Seq v4 Ultra Low Input RNA Kit (Clontech-Takara). Libraries were obtained and qualitatively assessed by using TapeStation 4200 and quantified by Qubit Fluorimeter. All samples were sequenced on an Illumina NextSeq 500 at an average of 40 million 75-bp single-end reads.
3'mRNA sequecing
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NextSeq 500 |
| |
|
| 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 (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 |
GPL19057 |
| Series (1) |
| GSE153975 |
Transcriptional profile of tumor associated macrophages in Ptenpc-/- and Ptenpc-/; Smad4pc-/- tumors |
|
| Relations |
| BioSample |
SAMN15468456 |
| SRA |
SRX8683544 |
