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Sample GSM7092431

Query DataSets for GSM7092431

Status

Public on Jun 26, 2024

Title

53371 (WT)

Sample type

SRA

Source name

53371

Organism

Mus musculus

Characteristics

cell line: 53371
cell type: Pancreatic Ductal Adenocarcinoma cell line
genotype: Wildtype

Treatment protocol

To generate isogenic cell lines that are deficient in JAK1, the parental lines were infected with pBabe-Cre-puro retroviral particles and selected with increasing concentrations of puromycin (2, 4, and 7 µg/ml).

Growth protocol

Mouse PDAC cells were maintained in DMEM medium supplemented with 10% FBS, L-glutamine, and nonessential amino acids as well as 10 µg/ml penicillin/ streptomycin and 50 µg/ml gentamicin.

Extracted molecule

total RNA

Extraction protocol

RNA was harvested using Rneasy mini plus kit (Qiagen). Total RNA was extracted from flash-frozen cultured cancer cells using the RNeasy Mini Kit (QIAGEN). QIAGEN's protocol was followed to extract the total RNA. The concentration of the RNA was determined on a NanoDrop spectrophotometer, and the integrity of the RNA was validated using gel electrophoresis. Quality and quantity of the total RNA was re-confirmed by Novogene. Only high quality RNA was used for library prepration. 1.3 ug of total RNA was used for the construction of sequencing libraries.
Library construction was performed by Novogene with the following procedure: After the QC, mRNA was enriched using oligo(dT) beads. Next, the mRNA was fragmented randomly by adding fragmentation buffer, followed by cDNA was synthesis using mRNA template and random hexamer primers. Next a custom second-strand synthesis buffer (Illumina) , dNTPs, RNase H and DNA polymerase I were added to initiate the second-strand synthesis. After a series of terminal repair, A ligation and sequencing adaptor ligation, the double-stranded cDNA library was completed through size selection and PCR enrichment. The quality of the library was confirmed via: (1) Qubit 2.0: tests the library concentration preliminarily. (2) Agilent 2100: tests the insert size. (3) Q-PCR: quantifies the library effective concentration precisely.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina NextSeq 500

Data processing

The quality of sequenced reads was evaluated using FastQC (v0.11.9).
For the differential expression analysis between parental and Jak1 knockout samples, the 75 or 150 base paired-end reads were aligned to the GRCm38/mm10 mouse reference genome (UCSC) via Rsubread (v2.0.0).
Transcript abundance was determined using the featureCounts function of the Rsburead package, and low abundance gene transcripts (cpm < 5 in more than half of the samples) were omitted in further downstream analyses.
The edgeR package was used for the paired sample differential expression analysis between wildtype parental cells and isogenic Jak1 KO cells. Genes that passed the threshold of an FDR (False Discovery Rate) below 0.05 were considered as significantly deregulated.
To visualize the differential expression of select deregulated genes, the gene counts were log2 transformed and scaled to highlight differences in isogenic cell line pairs and were subsequently plotted as a heatmap using the R-function heatmap.2 (package: gplots v3.0.3).
Assembly: mm10
Supplementary files format and content: Tab-delimited text files that contain gene Entrez IDs and corresponding normalized or normalized and log2 transformed values of gene counts for each sample.

Submission date

Mar 11, 2023

Last update date

Jun 26, 2024

Contact name

Kay-Uwe Wagner

E-mail(s)

wagnerk@karmanos.org

Organization name

Barbara Ann Karmanos Cancer Institute Wayne State University School of Medicine

Department

Department of Oncology