ABSTRACT: 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: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: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:The goal of the study was to compare gene expression of Robo1+/+ and Robo1-/- luminal progenitors. Total RNAs were then extracted from FACS purified luminal progenitor cells, harvested from Robo1+/+ or Robo1-/- mice (n=3 per genotype, two animals per n) using TRIreagent LS (Sigma, T3934). Poly(A)+ RNA sequencing libraries were made from each sample using the TruSeq RNA library preparation kit v.1 (Illumina). Illumina RNA PolyA library preparation guide. A total of 6 libraries were created by PCR amplification with Illumina barcoding primers using kit recommended conditions and quantified using a Bioanalyzer DNA 1000 kit (Agilent).
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: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.
Project description:Purpose: This study identifies the top transcripts expressed in mature erythrocytes and the top transcripts expressed in the polysome fractions of mature erythrocyte lysate Methods: RNA from total erythrocyte lysate and polysome fractions was isolated and purified using GeneJet RNA purification column (Thermo Fisher Scientific). The RNA library was prepared using NEBNext ULTRA II RNA library preparation kit for Illumina. The libraries were sequenced using Illumina Seq. Results: The transcriptome analysis revealed the abundance of mRNAs encoding globins genes, long non-coding RNAs, and micro RNAs. Deciphering the role and functions of these non-coding RNAs in these specialised cells could enable us to understand their biology in greater detail