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Global impact of CMP-001 on various immune cell subsets

ABSTRACT: Purpose: The goal of this study was to investigate the impact of CMP-001 on gene expression patterns within various immune cell subsets. Methods: Unfractionated PBMCs from a healthy donor were isolated via ficol gradient and treated with and without anti-Qbeta coated CMP-001 (10ug/ml) for 24 hours. Untreated and treated fractions were sequenced on an Illumina HiSeq4000 system. FASTQ files were generated from basecall files with the bcl2fastq software (Illumina) provided by the University of Iowa Institute of Human Genetics. Data files were subsequently downloaded to Argon and mapped to the prebuilt GRCh38 genome with CellRanger (version 3.0.1). After initial mapping datasets were merged together and clustering was performed based on merged data sets using Cell Ranger software. Cells with unique gene counts fewer than 300 or more than 4,000 per cell were eliminated along with a mitochondria gene cutoff of 25%. Log normalization of aggregated reads was performed with Seurat (version 3.0.2) using a scale factor of 10,000. Feature selection was performed with the variance stabilizing transformation before integration was performed with Seurat (v3.0.1). Results: A total of 17,280 cells were recovered after filtering. Clusters were identified based on expression of unique gene markers. Differential expression analysis identified genes enriched in each population of cells corresponding to untreated versus treated libraries. Particular emphasis was given to genes enriched in monocytes. Conclusions: We report that CMP-001 induces a variety of immune responses in each cell cluster, with a unique gene signature displayed by monocytes.

ORGANISM(S): Homo sapiens

PROVIDER: GSE162824 | GEO | 2021/06/30

REPOSITORIES: GEO

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Immune effects of CMP-001 vs. controls on various immune cell subsets

Project description:Purpose: The goal of this study was to investigate the TLR9 specific immune effects of anti-Qbeta-coated CMP-001 using the following groups: untreated, anti-Qbeta coated CMP-001, anti-Qbeta coated CMP-001 with methylated and thus, inactive ODN (mCMP-001), and G10 (naked TLR9 agonist). Methods: Unfractionated PBMCs from a healthy donor were isolated via ficol gradient and treated with and without anti-Qbeta coated CMP-001 (10ug/ml), anti-Qbeta coated mCMP-001 (inactive ODN; 10ug/ml), or G10 (naked TLR9 agonist; 2.5ug/ml) for 24 hours. Untreated and treated fractions were sequenced on an Illumina NovaSeq 6000 system. FASTQ files were generated from basecall files with the bcl2fastq software (Illumina) provided by the University of Iowa Institute of Human Genetics. Data files were subsequently downloaded to Argon and mapped to the prebuilt GRCh38 genome with CellRanger (version 3.0.1). After initial mapping datasets were merged together and clustering was performed based on merged data sets using Cell Ranger software. Cells with unique gene counts fewer than 300 or more than 4,000 per cell were eliminated along with a mitochondria gene cutoff of 25%. Log normalization of aggregated reads was performed with Seurat (version 3.0.2) using a scale factor of 10,000. Feature selection was performed with the variance stabilizing transformation before integration was performed with Seurat (v3.0.1). Results: A total of 17,280 cells were recovered after filtering. Clusters were identified based on expression of unique gene markers. Differential expression analysis identified genes enriched in each populatin of cells corresponding to untreated versus treated libraries. Particular empasis was given to genes enriched in monocytes. Conclusions: We report that CMP-001 induces a variety of immune responses in each cell cluster, with a unique gene signature displayed by monocytes.

2021-06-30 | GSE172493 | GEO

RNAseq analysis of treatment-resistant and sensitive breast cancer patient-derived organoids with paclitaxel treatments

Project description:Huntsman Cancer Institute (HCI)-001 patient-derived organoids (PDOs) and HCI-002 PDOs obtained from Huntsman Cancer Institute, University of Utah (Nat Med. 2011 Oct 23;17(11):1514-20). Purpose: The goals of this study is to identify molecular pathways and genes that are involved in the treatment responsiveness of breast cancer PDOs. Methods: mRNA profiles of HCI-001 and HCI-002 PDOs were generated by Illumina NovaSeq 6000 platform for paired-end sequencing, 150-bp read length, for about 20 million raw reads per sample. RNA sequencing FASTQ files were processed with HISAT2 aligner in default mode with the Ensembl human transcriptome annotation (Build version GRCh38 and transcript annotation GRCh38.89). Differential gene expression analysis was performed using DESeq2 software. Enrichment analysis was performed using ClusterProfiler software. Results: We mapped about 20 million sequence reads per sample to the human genome (build HG38) in the RNAs of HCI-001 and HCI-002 PDOs treated with paclitaxel or vehicle. We used a minimum expression cut-off of 1 FPKM in order to exclude low-levels genes and the RNA-seq data confirmed the difference expression of 67 known apoptosis-regulatory genes in treatment-sensitive PDOs (HCI-002) compare to treatment-resistant PDOs (HCI-001). Conclusions: This study identified apoptosis-regulatory genes that are differentially induced or represeed in treatment-sensitive PDOs (HCI-002) or treatment-resistant PDOs (HCI-001).

2021-09-09 | GSE183477 | GEO

RNA Sequencing analyses of control and HuD KO (Elavl4 null) mouse cortical transcriptomes

Project description:Purpose: The goal of this study was to analyse RNA-seq data to determine the effect of deletion of the RNA-binding protein HuD in transcriptiome-wide alternative splicing and polyadenylation in the neocortex of adult HuD KO vs. wild type littermates (controls) Methods: Cortical mRNA profiles of adult HuD KO (Elavl4 -/-) mice and Control mice were generated by RNA sequencing, in triplicate, using Illumina NovaSeq 6000 platform. The quality of raw RNA-sequencing reads was evaluated using FastQC software (version 0.11.5) and adapters were removed using the Cutadapt (version 1.15) and Trimmomatic (version 0.38) software. Alternative splicing was evaluated using rMATS software (version 4.0.2) and BAM files were converted to BedGraph before examining alternative polyadenylation using DaPars software (version 0.9.1) Methods (cont.): RNA-seq data was aligned to the M musculus genome (UCSC browser, mm10) using STAR (version 2.7.3a), and MultiQC (version 1.8) was used to perform a final quality check on STAR alignment files. If alignments were found to be the same read length and have >80% reads mapped to a unique location, the data was considered good quality and alternative splicing and polyadenylation analyses were performed. Sequence reads per sample were aligned to the mouse genome (build mm10). Results: HuD KO affected alternative splicing of 310 genes, including 17 validated HuD targets such as Cbx3, Cspp1, Snap25 and Gria2. In addition, deletion of HuD affected polyadenylation of 53 genes, with the majority of significantly altered mRNAs shifting towards usage of the proximal polyadenylation signal (PAS), resulting in shorter 3’ untranslated regions (3’ UTRs). Conclusions: HuD KO had a greater effect on alternative splicing than polyadenylation, with many of the affected genes implicated in several neuronal functions and neuropsychiatric disorders.

2021-03-17 | GSE169023 | GEO

Broad and largely overlapping molecular changes arise in thymic and peripheral XCR1+ dendritic cells upon tolerogenic and immunogenic maturation

Project description:This SuperSeries is composed of the SubSeries listed below. DC were isolated from lymphoid organs as previously described (Vremec et al., 2000). DC subsets were sorted by flow cytometry according to the marker combinations described in the 'characteristics: phenotype' field for each sample for each individual series. GeneChip Mouse Gene 1.0 ST (GPL6246) and GeneChip Mouse Genome 430 2.0 (GPL1261) DAT files were processed using Affymetrix Gene Chip Operating System to generate .CEL files. Normalization of the raw Affymetrix expression data with Robust Multi-chip Analysis were performed through Bioconductor in the R statistical environment (version 3.0.2) using the oligo package (GPL6246) and the affy, affyPLM and yaqcaffy packages (GPL1261). For genes symbol with several probeSets, the most variant probSet has been selected. The two platforms where merged by the unique genes symbols. Quantile Normalization was applied on the merged expression arrays to generate Matrix 1. PCA analysis revealed a strong platform effect. Partial Least Square (PLS) was performed to remove the platform effect and yield Matrix2 which was used for subsequent analyses. The data matrices contain log2 values.

2016-08-22 | E-GEOD-71171 | biostudies-arrayexpress

Long ncRNA in HCC cells

Project description:Normal human hepatocytes and 3 different HCC cell lines have been analyzed for the global expression of long non coding RNA (lncRNA). Total RNA was extracted from each sample using RNeasy Kit (Qiagen) according to the manufacturer’s instructions. RNA quality was assessed by Nanodrop ND-1000 and Bioanalyser2100. DNA microarray The Human Long Non-coding RNA Array was manufactured by Roche NimbleGen. Each array represents all long transcripts, both protein coding mRNAs and lncRNAs (long non-coding RNAs) in the human genome. More than 22000 lncRNAs are collected from the authoritative data sources including NCBI RefSeq, UCSC, RNAdb, lncRNAs from literatures and UCRs. Fluorescent cDNA targets were prepared using NimbleGen One-Color DNA Labeling Kit. Microarray hybridization was performed at 42°C for 16-20 hours in NimbleGen Hybridization System. After being washed in an ozone-free environment, the slides were scanned using the Axon GenePix 4000B microarray scanner. Data Analysis Raw data were extracted as pair files by NimbleScan software (version 2.5). NimbleScan software’s implementation of RMA offers quantile normalization and background correction. The Probe level (*_norm_RMA.pair) files and Gene summary (*_RMA.calls) files were produced. The gene summary files were imported into Agilent GeneSpring Software (version 10.0) for further analysis. Differentially expressed genes and non-coding RNA were identified through Fold-change screening. Unsupervised hierarchical clustering, GO analysis and Pathway analysis was performed using the Agilent GeneSpring GX software (version 10.0).

2013-12-01 | GSE25859 | GEO

Expression data from G0 versus G4/G5 CMP/GMP

Project description:Telomere dysfunctional CMP/GMP have deregulated pathways that are associated with DNA damage signaling We compared differentially expressed genes in the G4/G5 CMP relative to the G0 control to identify pathways that may affect CMP differentiation. Bone marrow CMP and GMP cells were sorted from two paired pools of G0 TERTER/+ or G4/G5 TERTER/ER mice (5,000-20,000 cells per sample) using the Influx Cell Sorter. Every paired pool includes CMP or GMP sorted from 4 age and gender matched G0 or G4/G5 mice. RNA from the respective sorted cells was extracted using Trizol (Ambion) and profiled on 2100 Bioanalyzer (Agilent). Gene expression profiling was performed at the Sequencing and Non-coding RNA Program at MD Anderson Cancer Center. Briefly, the GeneChip® 3 IVT Express Kit (Affymetrix) was used to generate biotin-labeled cRNA, which were purified and fragmented, before target hybridization on the GeneChip® Mouse Genome 430 2.0 Array (Affymetrix) according to the manufacturer's instructions. Affymetrix raw data (CEL files) were normalized using Affymetrix Microarray Suite (MAS) version 5.0 using a TGT=100. Paired pools used in the study were: CMP pool 1: G0-1 and G4/5-1 CMP pool 2: G0-2 and G4/G5-2 GMP pool 1: G0-1 and G4/G5-1 GMP pool 2: G0-2 and G4/G5-2

2015-04-09 | E-GEOD-62278 | biostudies-arrayexpress

NimbleGen Rat Gene Expression Microarrays for 2 samples (icariin/DMSO-treated BMSC)

Project description:Total RNA from each sample was quantified by the NanoDrop ND-1000 and RNA integrity was assessed by standard denaturing agarose gel electrophoresis. Total RNA of each sample was used for labeling and array hybridization as the following steps: 1) Reverse transcription with by Invitrogen Superscript ds-cDNA synthesis kit; 2) ds-cDNA labeling with NimbleGen one-color DNA labeling kit; 3) Array hybridization using the NimbleGen Hybridization System and followed by washing with the NimbleGen wash buffer kit; 4) Array scanning using the Axon GenePix 4000B microarray scanner (Molecular Devices Corporation). Scanned images (TIFF format) were then imported into NimbleScan software (version 2.5) for grid alignment and expression data analysis. Expression data were normalized through quantile normalization and the Robust Multichip Average (RMA) algorithm included in the NimbleScan software. The Probe level (*_norm_RMA.pair) files and Gene level (*_RMA.calls) files were generated after normalization. The 2 gene level files were imported into Agilent GeneSpring GX software (version 11.5.1). Genes that have values greater than or equal to lower cut-off: 100.0 in that at least 1 out of 2 samples (“All Targets Value”) were chosen for data analysis.

2022-07-20 | GSE208583 | GEO

Analysis of single-cell RNA-seq data from human PBMCs and from in vitro cultures of human cord blood CD34+ progenitors encompassing different DC types

Project description:For both PBMC and cells from the in vitro cultures, RNA purification and library generation was performed using the Chromium Single Cell Controller apparatus and associated protocols (10X Genomics). Libraries were sequenced by 75-bp single-end reading on a NextSeq500 sequencer (Illumina). Reads were aligned on the GRCh38 human genome assembly. Data analysis was performed using the R software package Seurat (https://github.com/satijalab/seurat)

2018-08-14 | GSE115405 | GEO

Combined Immunotherapy and Radiosurgery for Metastatic Colorectal Cancer

Project description:A single institution study to evaluate the safety and tolerability of the combination treatment of nivolumab, ipilimumab, CMP-001 and radiosurgery in patients with metastatic colorectal cancer with liver metastases.

| 2271587 | ecrin-mdr-crc

High resolution single cell transcriptomics reveals heterogeneity of self-renewing hair follicle stem cells

Project description:Purpose: To dissect the transcriptomic profiles and unravel population-specific transcriptional heterogeneity of self renewing hair follicle stem cells in vivo Methods: We performed 10x genomics single-cell RNA sequencing (scRNA-seq) of FACS sorted CD34+/K14-H2BGFP+ hair follicle stem cells from mouse skin at mid-anagen. FACS purified CD34+/K14-H2BGFP+ single-cell suspension was processed for the barcoded single-cell 3′ cDNA libraries generation using Chromium Single Cell 3′ gel bead and library Kit v3. The final libraries were quantified using Agilent Bioanalyzer high sensitivity DNA chip and sequenced using an Illumina NextSeq-500. The raw data files were demultiplexed to generate the sample-specific FASTQ files, which were aligned to the mouse reference genome (mm10-3.0.0) using the 10x Genomics Cell Ranger pipeline (v3.1.0). The raw scRNA-seq data was processed using Cell Ranger from the 10x platform to generate an expression matrix that was further analyzed in R using the Seurat package version 3.1. Only high-quality cells that had between 200 and 5000 genes expressed and had under 10% of the UMIs mapped to mitochondrial genes were retained. Results: We obtained a total of 6736 high quality cells from two datasets for further analysis by Seurat workflow Conclusions: Obtained high quality single cell transcriptomic data to dissect molecular heterogeneity of hair follicle stem cells

2021-04-14 | GSE162333 | GEO

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