Adipocyte-driven unfolded protein response is a shared transcriptomic signature of metastatic prostate carcinoma cells
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ABSTRACT: Purpose: To begin understanding molecular processes that enable tumor cells to survive and progress in difficult microenvironments such as bone, we performed unbiased examination of the transcriptome of two different prostate cancer cell lines in the absence or presence of bone marrow adipocytes. Methods: 3’ RNA-seq (QuantSeq 3′ mRNA) was performed. RNA from four biological replicates of PC3 and ARCaP(M) cells cultured alone or in Transwell with adipocytes was collected , and run on an Agilent TapeStation 2200 (Agilent Technologies, Santa Clara, CA) for quality control. Lexogen’s QuantSeq 3’mRNA-seq Library Prep Kit (FWD for Illumina) was utilized for building RNAseq libraries. The barcoded libraries were multiplexed at equimolar concentrations and sequenced with 50 bp single reads on an Illumina HiSeq-2500 run in rapid mode. Data were demultiplexed using Illumina's CASAVA 1.8.2 software and reads were aligned to the human genomes. Differential gene expression analysis was used to detect adipocyte-mediated transcriptome changes. qRT–PCR validation was performed using TaqMan assays. Results: Our RNAseq analyses and subsequent quantitative PCR and protein-based assays reveal that upregulation of Endoplasmic Reticulum (ER) stress and Unfolded Protein Response (UPR) genes is a shared signature between two metastatic prostate carcinoma cell lines of different origin. Pathway analyses and pharmacological examinations highlight the ER chaperone BIP as an upstream coordinator of this transcriptomic signature. Conclusions: Our studies pave the way for additional mechanistic investigations and offer new clues towards effective therapeutic interventions in metastatic disease.
Project description:The goal of this study is to use RNA-seq to investigate the molecular mechanisms by which REST inhibition regulates corticalspinal axon regeneration. To achieve this goal, cortical motor neurons from adult wild-type (WT) and REST conditional knockout (cKO) were purified at 1, 3, 7 days following spinal cord injury (SCI) or sham operation. RNA-seq libraries for cortical motor neurons were prepared with the QuantSeq mRNA-Seq library prep kit FWD for Illumina (Lexogen) and sequenced using HiSeq4000 (Illumina). Sequenced reads were mapped to reference genome (mm10) using STAR, and count level data were quantifed using Salmon. Differential gene expression analysis was performed using limma voom.
Project description:The goal of this study is to compare mRNA expression profiles among wild type, MBD2 knock down, p66α knock down, and ABA- and APC-treated cells.. All cell lines were maintained in DMEM supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin. For the doxycycline inducible gene expression regulation, each cell lines were incubated in the presence of 1 μg/ml doxycycline for 2 days. For the drug treatment, MDA-MB-231 (LM1) cells were incubated in the presence of 10 μM ABA or APC for 2 days. Total cellular RNAs were extracted using Qiazol reagent. The RNA quality was examined by spectrophotometry, agarose gel electrophoresis (calculating the 18S and 28S rRNA ratio) and an Agilent Technologies 2100 Bioanalyzer (ensuring a RIN value greater than 7). Library was prepared with QuantSeq 3’ mRNA-Seq Library Prep Kit FWD. The constructed libraries were subjected to 75-bp single-end sequencing using an Illumina NextSeq 500 sequencer. Using an optimized data analysis workflow, we mapped to the human genome (build hg19) and identified 25,737 transcripts in these cell lines.
Project description:3' mRNA-seq was performed with QuantSeq 3' mRNA-Seq kit (Lexogen 015) according to the manufacturer’s recommendations. 3' mRNA-seq was done in biological triplicates (soma) or duplicates (neurites), using 260 ng of total RNA from neurites or soma of mESC-derived neurons per sample. Libraries were pooled and sequenced on Illumina NextSeq 500 system with a single-end 150-cycle run.
Project description:Freshly dissected Thymus, Kidney, and Spleen from young and aged mice were subjected to collagenase digestion to prepare single-cell suspension for the isolation of endothelial cells. Endothelial cells form the single-cell suspension was isolated using a magnetic bead-based separation method using CD144 and Endomucin antibody. RNA from 150,000 cells from each group was isolated using the RNeasy Plus Micro Kit (QIAGEN) according to the manufacturer’s instructions. The library preparation was performed using QuantSeq 3’ mRNA-Seq Library Prep Kit FWD for Illumina.
Project description:To assess how LARP6 affects mRNA localisation to cell-protrusions, we transfected MDA-MB231 cells with either non-targeting control siRNA or LARP6 siRNA for 72 hrs, before fractionating the cells into protrusion and cell-bodies, and analysed total RNA from each fraction via Lexogen QUANTSEQ FWD 3' mRNA sequencing. Protrusion induction was done for 2 hrs on 3 micron transwell filters (Corning). The experiment was performed twice using two independent batches of cells, and sequencing was carried out on an Illumina NextSeq 500 sequencer.
Project description:<p>Exome sequencing of matched pairs of tumor / normal genomic DNA was performed from high risk localized prostate cancer or lethal, metastatic, castrate resistant prostate cancer (CRPC). Exome libraries were prepared using Illumina Paired_End Genome DNA Sample Prep Kit and captured using Agilent SureSelect Capture Library or Roche EZ Exome capture library. Sequencing was performed on Illumina GAII and HiSeq 2000 platforms in paired end mde, with 80 base pair reads from the final library fragments. Copy number alterations and somatic mutations were identified.</p>
Project description:3' mRNA-Seq obtained from distinct isolated cell types (epithelia cells,immune cells, fibroblasts) of endoscopically obtained esophageal adenocarcinoma tissue as well as normal esophageal mucosa. Libraries for RNA-sequencing were prepared using the QuantSeq 3' mRNA-Seq Library Prep Kit FWD for Illumina according to the low input protocol. Libraries were sequenced on a HiSeq 4000 (Illumina) by 1x 50 bases.
Project description:Spheroids, near spherical multicellular aggregates, are one of the most common types of threedimensional (3D) cell cultures. Despite decades of implementation of spheroid technology in various fields of life science and medical research, no minimal information (MI) guidelines are available to cope with heterogeneity and to stimulate transparency. To cope with this unmet need, we assembled an international consortium to develop the MISpheroID knowledgebase (https://www.mispheroid.org) and interrogation revealed heterogeneity and lack of transparency in published spheroid-related experiments. This steered us to empirically evaluate the impact of cell line, culture medium type, spheroid formation method and spheroid size on complementary spheroid metrics (RNA fingerprints, presence of cell death, ATP content, glucose to lactate conversion, secreted protein signatures, circularity, size and cancer therapy response). We measured media-induced transcriptional variation in lung cancer (A549), colorectal cancer (HCT116), ovarium cancer (SKOV3) and glioblastoma (U87MG) spheroids using RNA sequencing (RNA-seq). These spheroids were formed in ultra-low attachment plates and cultured in 6 different media types (DMEM high glucose, DMEM/F12, RPMI1640, DMEM low glucose, EMEM and MEM) for 5 (HCT116) or 7 (A549, SKOV3 and U87MG) days. RNA extraction was performed on 2 spheroids per condition using the miRNeasy micro kit (217084, Qiagen, Hilden, Germany). RNA-sequencing libraries were prepared from purified RNA using the QuantSeq 3' mRNA-Seq Library Prep Kit FWD for Illumina (Lexogen, Vienna, Austria) according to the manufacturer's instructions, using 27.5ng of RNA that was DNase treated using HL-dsDNA (Arcticzymes, TromsØ, Norway). The individual libraries were quantified by qPCR using the KAPA Library Quantification Kit (Roche, Pleasanton, CA, US) and equimolarly pooled. The pool concentration was measured with Qubit and 1.4pM with 1% PhiX was sequenced on a NextSeq 500 (Illumina, San Diego, CA, US) using a high-output 1x75 run. Reads were mapped to the human genome using Tophat and gene expression counts were generated using HTSeq. This data was used to perform Principal Component Analysis (PCA) and Gene Set Enrichment Analysis (GSEA).
Project description:Purpose: To investigate mechanisms underlying SPANX knockdown phenotypes, we monitored changes in gene expression profiles in A375 melanoma cells subjected to SPANX KD (RNA-Seq). Methods: RNASeq Libraries were prepared from isolated total RNA using the QuantSeq 3' mRNA-Seq Library Prep Kit FWD for Illumina from Lexogen, (Vienna, Austria). Barcoded libraries were pooled and single end sequenced (1X75) on the Illumina NextSeq 500 using the High output V2.5 kit (Illumina Inc., San Diego CA). Raw data FASTQ files were generated by standstard illumina base calling pipeline and stored in illumina cloud service BaseSpace. Reads were aligned with the STASR aligner. Analyses of differentially expressed genes (DEGs) were subsequently performed using a negative binomial test method (edgeR)10 implemented using SARTools R Package Results: Of 609 genes deregulated by both shRNA, 454 were upregulated and 123 were downregulated. Networks associated with cell division were downregulated following SPANX KD, while those associated with 3-phosphoinositide biosynthesis were upregulated. Notably, IPA analysis identified the cell cycle as the primary pathway deregulated, and further analysis using the reactome database revealed that most of those genes were associated with the G1/S transition.