ABSTRACT: Control untransduced or anti-HER2 CAR (Ad5f35 transduced) human macrophages were exposed to control media, human IL-4, or human IL-13, and the response to these cytokines was measured at a transcriptome-wide level. RNA was harvested 24 hours post cytokine exposure with Ambion RiboPure RNA Purification Kit (Thermo Fisher) and RNA-Seq libraries were generated using TruSeq RNA Library Prep Kit (Illumina). Libraries were sequenced on 75bp single-end reads using a NextSeq (Illumina).
Project description:Human monocyte derived macrophages (GM-CSF differentiated) were stimulated with control media, IFNy/LPS (M1), IL4 (M2), empty vector Ad5f35 (MOI 1000), or anti-HER2 CAR Ad5f35 (MOI 1000). RNA was harvested 48 hours post transduction with Ambion RiboPure RNA Purification Kit (Thermo Fisher) and RNA-Seq libraries were generated using TruSeq RNA Library Prep Kit (Illumina). Libraries were sequenced on 75bp single-end reads using a NextSeq (Illumina).
Project description:The blood samples of PD patients and Healthy controls were collected from the general OPD of Department of Neurology, KGMU, Lucknow. RNA (including small RNAs) was extracted by Ribopure Blood RNA Isolation Kit by Thermo Fisher as suggested protocol by manufacturer. A custom Taqman OpenArray panel was used to profile the miRNAs expression.
Project description:12 week male wistar rats were exposed with rotenone (2.5mg/kg b.wt.) and control rats exposed with vehicle (corn oil) intraperetonially for two months. Animals were sacrifised at the end of the experiment and blood was isolated and stored at -80°C in RNALater until further analysis.RNA (including small RNAs) was extracted by Ribopure Blood RNA Isolation Kit by Thermo Fisher as suggested protocol by manufacturer. A custom Taqman OpenArray panel was used to profile the miRNAs expression.
Project description:<p>To determine IL-17-induced global transcriptome changes in midbrain neurons derived from induced pluripotent stem cells (iPSC) from three sporadic Parkinson's disease (PD) patients and three age- and sex-machted controls, deep RNA sequencing (RNA-Seq) of IL-17-treated and untreated PD and control iPSC-dderived midbrain neurons was performed. Total RNA was isolated from untreated and IL-17-treated cells with the TruSeq RNA Sample Preparation Kit v2 (Illumina). RNA libraries were quantified using the KAPA SYBR FAST ABI Prism Library Quantification Kit (Kapa Biosystems) and cluster generation was performed on the cBot with the TruSeq SR Cluster Kit v3 (Illumina). The sequencing run was performed on a HiSeq 1000 instrument (Illumina) using the indexed, 50 cycles single read (SR) protocol and the TruSeq SBS v3 Kit (Illumina). Image analysis and base calling resulted in .bcl files that were then converted into .fastQ files by the CASAVA1.8.2 software. FastQ files were aligned to the human genome (hg19) using STAR.and annotated with gencode.v19. DESeq2 was used to determine differential expression. Criteria to determine significantly dysregulated genes were as follows: adjusted p-value below 0.05 and log2FC (fold change) of greater than one. Only genes with a mean expression value of greater than one RPKM (reads per kilobase per million mapped reads) throughout the dataset were considered. Control and PD samples were analyzed as two independent datasets.</p> <p>Upon IL-17 treatment only 17 genes were found to be dysregulated in controls but 125 genes were dysregulated in iPSC-derived midbrain neurons from PD patients. The 125 IL-17-dependent genes in PD iPSC-derived neurons separated the treated from untreated PD samples using an unsupervised, hierarchical clustering applying an Euclidean distance metric.</p> <p>More detailed study information can be found in Sommer A, Maxreiter F, Krach F, Fadler T, Grosch J, Maroni M, Graef D, Eberhardt E, Riemenschneider MJ, Yeo GW, Kohl Z, Xiang W, Gage FH, Winkler J, Prots I, Winner B. Th17 Lymphocytes Induce Neuronal Cell Death in a Human iPSC-Based Model of Parkinson's Disease. Cell Stem Cell. 2018 Jul 5;23(1):123-131.e6. doi: 10.1016/j.stem.2018.06.015. PMID: 29979986</p>
Project description:Purpose: RNA sequencing across whole genome downstream to IL-1 to investigate scope and nature of effect of IFT88 depletion Method: Control pool treated and IFT88 siRNA pool targeted Fibroblast-like chondrocytes were treated with or without IL-1B 10ng/ml and RNA collected in triplicate at 0, 1, 2, 4, 8 and 24h. PolyA-selected sequencing libraries were prepared using the TruSeq protocol (Illumina). Libraries were subject to 75bp paired-end sequencing (Illumina HiSeq 4000) to an average depth of 28.5 million read pairs per sample and aligned to mouse genome with Hisat2. Results: Using time-course analysis (ImpulseDE2) the IL-1 response timecourse was determined (control-only) and intersected against the comparative case vs control analysis. Per-point differential expression analysis IFT88 vs control was performed using DESeq2. Conclusion: Demonstration of the effect of IFT88 depletion on IL-1 response signature over 24h.
Project description:Purpose: The study aimed to characterize the molecular phenotype of bone marrow macrophages in NHL with RNA sequencing analysis. Methods:RNA of sorted femur macrophages (CD11b+F4/80+) were extracted with an RNA extraction kit (Qiagen). The samples were submitted to Novogene Inc. for library preparation and subsequent RNA sequencing. RNA was used for cDNA library construction using an NEBNext® Ultra RNA Library Prep Kit for Illumina® (New England Biolabs) according to the manufacturer’s protocol. The resulting 250-350 bp insert libraries were quantified using a Qubit 2.0 fluorometer (Thermo Fisher Scientific) and quantitative PCR. Size distribution was analyzed using an Agilent 2100 Bioanalyzer (Agilent Technologies). Qualified libraries were sequenced on an Illumina HiSeq 4000 Platform (Illumina) using a paired-end 150 run (2×150 bases). Reads containing adapter or poly-N and those of low quality were trimmed, after which gene counts were obtained though mapping the clean reads to reference genome mm10 using STAR 2.5.3a.
Project description:We employed miCLIP-seq to profile the location and extent of m6A in the Poly(A)+ transcriptome. Skin epithelial cells were isolated from wild-type P0 neonates by FACS. Total RNA was extracted by TRIzol-LS and Poly(A)+ RNA was extracted with Dynabeads™ mRNA Purification Kit (Thermo-Fisher, 61006). Input and miCLIP libraries was prepared from 3 biological replicates with each containing RNA isolated from 3 litters of neonates. Libraries were sequenced on the Illumina Hi-seq platform to generate paired-ended 50 bp reads. Sequencing data was processed as described in (Geula et al., 2015).
Project description:Valproate(VPA) has been used in the treatment of bipolar disorder since the 1990s. However, the therapeutic targetsof VPA have remained elusive. Here we used RNA sequencing in human iPSCs derived from bipolar patients to further identify important molecular targets. Human iPSCs were homogenized and total RNA was isolated using the RNeasy Plus Micro Kit (Qiagen, Hilden, Germany). RNA quantity and quality were assessed using fluorometry (Qubit RNA Broad Range Assay Kit and Fluorometer; Invitrogen, Carlsbad, CA) and chromatography (Bioanalyzer and RNA 6000 Nano Kit; Agilent, Santa Clara, CA), respectively. Libraries were prepared using TruSeq Stranded mRNA (PolyA+) kit (Illumina, San Diego, CA) and sequenced by Illumina NextSeq 500. The read length was 75bp with 30-40M reads per sample. FastQC (v0.11.3) was performed to assess data quality. TopHat2 (v2.0.9) aligned the reads to the mouse reference genome (Mus musculus UCSC mm10) and to the Ensembl human reference genome (GRCh38.p13) using default parameters. Alignments were then converted to expression count data using HTseq (v0.6.1) with default union mode.
Project description:To determine transcriptomic changes in cellular targets induced by MCV-miR-M1. Briefly, HEK293 cells were transfected with either MCV-miR-M1-5p, MCV-miR-M1-3p or control mimic (Thermo Fisher Scientific) prior to RNA extraction and confirmation of MCV miRNA 5p and 3p expression via stem loop qRT-PCR. Total RNA libraries were prepared using TruSeq Stranded Total RNA Sample Prep Kit (Illumina, USA) and the TruSeq cDNA libraries were analysed via Illumina HiSeq2500 paired end 100bp.
Project description:Purpose: This study is designed to understand whether Epidermal Growth Factor treatment alters the transcriptome of hematopoietic stem cells after radiation injury Method: RNA was extracted from C57BL/6J mice at six weeks post 500cGy total body radiation and Epidermal Growth Factor or saline subcutaneous treatment. Libraries for RNA-Seq were prepared with Clontech kit. The data was sequenced on NextSeq500 for a paired-end 75bp read run. Data quality check was done on Illumina SAV. Demultiplexing was performed with Illumina Bcl2fastq2 v 2.17 program. The Partek flow and Ingenuity Pathway Analysis (IPA) were used for bioinformatics methods and data analysis, respectively. The reads were mapped to the latest UCSC transcript set using STAR - 2.7.2a and GRCm38.97.