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Single-cell full-length RNA-sequencing of MCF7 and BT-474 cells using down-scaled Smart-seq2

ABSTRACT: We report single-cell RNA-sequencing data of wild type, breast cancer cells MCF7 and BT-474. Single cells were isolated using the F.SIGHT single cell dispenser. cNDA synthesis and library preparation were down-scaled to 1/10 of original reaction volumes. By comparative UMAP clustering we could show that our MCF7-cluster exactly overlapped with sequencing data from GSE151334. Data was analyzed using three common alignment tools: salmon, kallisto and STAR. DE analysis was performed with inputs from each aligner. We proved the overexpression of ErbB2 in BT-474 compared to MCF7 in all cases.

ORGANISM(S): Homo sapiens

PROVIDER: GSE201443 | GEO | 2023/12/31

REPOSITORIES: GEO

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Project description:Background: Neuropilin-1 (NRP-1) is a non-tyrosine kinase glycoprotein receptor that elicits multiple functions depending on the ligand bound. It is known to promote cancer progression however; transcriptome-wide changes triggered by NRP-1 are not defined. In this study we aimed to identify novel molecules associated with NRP-1 expression using a global transcriptomic approach in a recombinant breast cancer cell model. Methods: NRP-1 was stably overexpressed in the BT-474 breast cancer cell line (BT-474 NRP-1) and subjected to next generation RNA sequencing using the Illumina HiSeq 4000 sequencing system. Two replicates each of the control BT-474 and BT474-NRP1 were utilized for sequencing. The count per million (CPM) method was utilized for filtering low counts/noise by NOISeq. The clean reads were mapped to reference genome using HISAT/ Bowtie2 tool. The Fragments Per Kilobase of transcript per Million mapped reads (FPKM) method was utilized to calculate the expression levels. False Detection Rate (FDR) ≤ 0.001, absolute value of Log2 ratio ≥ 2 and consistency between the two replicates were used as the default threshold to identify significant DEGs. Results: A total of 22,655,647 clean reads from 22,914,684 raw sequencing reads were generated upon sequencing. 22 upregulated and 61 downregulated genes were identified. Among the DEGs, 11 upregulated and 11 downregulated genes were shortlisted based on their known roles in cancer and/or EMT for confirmation with real-time qPCR. 2 upregulated genes TNC and TARP and 5 downregulated genes ACE, APOD, DDIT3, P2RX6 and ATF3 indicated consistently differential expression with qPCR. Conclusion: We report for the first time the global transcriptome-wide changes elicited by NRP-1 overexpression in a breast cancer cell model. Our study identified the novel association between several candidate molecules such as TNC, a known oncogene, and NRP-1 expression that may controbute to the tumourigenic role of NRP-1 in breast cancer.

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Single-cell full-length RNA-sequencing of MCF7 and BT-474 cells using down-scaled Smart-seq2

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