Next Generation Sequencing analyses negative control (NC) and TRPC5OS overexpressed MDA-MB-231 Transcriptomes
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ABSTRACT: Purpose: Next-generation sequencing (NGS) has revolutionized systems-based analysis of cellular pathways. The goals of this study are to compare the differential genes between negative control and TRPC5OS overexpressed MDA-MB-231. Method: RNA was extracted as aforementioned from cells following after TRPC5OS was overexpression.sed. Agarose gel electrophoresis was used for assessing the integrity testingof the extracted RNAs,, and quantification and further quality tests were performed using a NanoDrop ND-1000 (NanoDrop Technologies, Wilmington, DE, USA). Subsequently, an NEB Next ®Poly (A) mRNA Magnetic Isolation Module was used for mRNA enrichment by adding it into the total RNA extract. The library of the processed RNA product RNA was established using the KAPA Stranded RNA-Seq Library Prep Kkit (Illumina, Inc., San Diego, CA, USA). The established library was sequenced using the Illumina HiSeq 4000 platform. Conclusions: The results showed that total 55,256 genes were dysregulateddifferentially expressed, including 3,269 upregulated genes and 1,987 downregulated genes (fold change ≥1.5) .
Project description:Purpose: Next-generation sequencing was carried out to characterize transcripts landscape of the Microplast derived from the MCF7 cells treated with macrophage conditioned medium. Methods: To stimulate microplasts formation, MCF-7 cells were cultured in 50% macrophage conditioned medium and 50% DMEM growth medium for 48 h. Cells and microplasts were harvested and resuspended in ice cold PBS. A low-speed differential centrifugation protocol was used to isolate microplasts. Total RNA was extracted from two biological replicates of microplasts enriched samples using RNeasy mini kit (Qiagen, Hilden, Germany). RNA concentration and integrity were determined with Nanodrop 1000 (Nanodrop, Wilmington, DE,USA) spectrophotometry. Libraries were prepared using the TruSeq RNA sample prep kit V2 as per the manufacturer’s protocol (Illumina, San Diego, USA). Conclusions: This study provide the first transcriptional profile of the microplast derived from the MCF7 cells.
Project description:Illumina technology was used to generate mRNA profiles of stem apex of Populus yunnanensis with cutting and inverted cutting. Total RNA was extracted separately from each plant and pooled to three biological replicates per condition. RNA concentration and purity was measured using NanoDrop 2000(Thermo Fisher Scientific, Wilmington, DE). RNA integrity was assessed using the RNA Nano 6000 Assay Kit of the Agilent Bioanalyzer 2100 system (Agilent Technologies, CA, USA).
Project description:RNA was isolated from control and FTO,METTL3 deficient mouse 3T3-L1 cells using the TRIzol (Invitrogen) reagent by following the company manual.Total RNA was isolated from transiently transfected cells with TRI® Reagent (Sigma). mRNA was extracted using biotinylated poly(dT) oligo, followed by further removing of contaminated rRNA using RiboMinus Transcriptome Isolation Kit (K1550-02, Invitrogen). mRNA quality was analyzed by NanoDrop. Approximately 2.5 µg of total RNA was then used for library preparation using a TruSeq™ RNA Sample Prep Kit v2 (Illumina, San Diego, CA, USA) according to the manufacturer’s protocol.The libraries were sequenced using HiSeq2000 (Illumina) in single-read mode, creating reads with a length of 101 bp. Sequencing chemistry v2 (Illumina) was used and samples were multiplexed in two samples per lane.
Project description:Purpose:The goal of this study is to compare the miRNA expressions in LPS stimulated RA FLSs derived EVs between trauma FLSs derived EVs. Methods:Total RNA of EVs was extracted with trizol LS (Invitrogen, USA), and quantified with Nanodrop. Agarose electrophoresis was used for quality inspection, and library was constructed after passing the quality inspection. Then, Agilent 2100 Bioanalyzer was used to determine the quality of the library. RNA was denatured with 0.1 M NaOH and sequenced on Illumina NextSeq500. Results:In total of 24 miRNAs were changed in the 650 scanned miRNAs in LPS stimulated RA FLSs derived EVs, compared to Trauma FLSs derived EVs, with 13 up-regulated miRNAs and 11 down-regulated miRNAs(P<0.05; Fold change>=1.5).Next, we analyzed the possible biological functions of the expression changed miRNAs using GO analysis. Of note, the results showed that expression altered miRNAs may principally correlate with blood vessel morphogenesis, cardiovascular system development, vasculature development and tube development. Conclusions:RNA parts of LPS-RA FLSs derived EVs may play key role in stimulating angiogenesis of ECs.
Project description:We report the differential abundance of cell free miRNAs extracted from extracellular vesicles (EV) in the peripheral blood of pregnant women with or without preeclampsia by Next Generation Sequencing. Maternal blood samples were collected form pregnant women enrolled in the study during first to very early second trimester (11–14 weeks ), mid- to late second trimester (19–22 weeks), third trimester (36 weeks) and at delivery. Extracellular vesicles were isolated from peripheral blood and total RNA was extracted using the miRNeasy Mini Kit (Qiagen, Valencia, CA) following manufacturer’s instruction. miRNA libraries were prepared utilizing NEB Next Multiplex small RNA Library prep kit (NEB E7300S; New England Biolabs, Inc, Ipswich, MA, USA) and libraries were subsequently sequenced using the HiSeq-2500 platform with single-end 50bp reads (Illumina Inc.; San Diego, CA, USA).
Project description:For the bilateral intrastriatal delivery of AAV expressing control or TRAX shRNA, mice were anesthetized with ketamine/xylazine HCl (87.56 and 7.5 ug/kg, respectively, via an intraperitoneal injection) and injected with the indicated AAV (2 µl per injection spot, 2.5*10^9 vector genome/µl) with a 10 ml syringe (Hamilton, Reno, NV, USA) at a rate of 0.5 ul/min. The AAV was injected at the desired position: AP+0.5, L±2, DV-2.5 and -3.5 mm relative to bregma and the dura surface. Then the mouse brains were removed from the mice directly for dissection of striatum. RNA was extracted by TRIzol reagent or miRNeasy mini kit (Qiagen). DNase digestion was performed by the DNA-free™ Kit (Life Technologies, Carlsbad, CA, USA) or the RNase-free DNase set (Qiagen) following the manufacturer’s instructions. The concentration and purity of RNA were measured at 260, 280 and 230 nm by a NanoDrop (Thermo Fisher Scientific, Waltham, MA, USA). RNA was qualified by a Bioanalyzer 2100 (Agilent Technology, Santa Clara, CA, USA) with an RNA 6000 LabChip kit (Agilent Technology). For mRNA, the RNA was prepared based on Illumina’s official protocol. Library construction was carried out by TruSeq Stranded Total RNA Library Prep Gold Kit (Illumina, San Diego, CA, USA) followed by AMPure XP beads (Beckman Coulter, Brea, CA, USA) size selection. Libraries were sequenced using sequencing-by-synthesis (SBS) technology (Illumina). Sequencing data and FASTQ reads were generated using an inhouse Welgene ’Biotech pipeline according to Illumina’s base calling program bcl2fastq v2.20.
Project description:We performed RNA-seq experiments (three replicates) on primary mouse podocytes that were infected with control lentivirus or KDM6A-encoding lentiviruses. RNAs were first extracted using Trizol reagent (Invitrogen, USA) according to the manufacturer’s instruction. The purified RNAs were quantified at OD260nm using a ND-1000 spectrophotometer (Nanodrop Technology, USA) and qualified using a Bioanalyzer 2100 (Agilent Technology, USA) with RNA 6000 LabChip kit (Agilent Technology, USA). All procedures for library preparation and sequencing were performed according to the Illumina protocol. Library construction was carried out using the TruSeq RNA Library Preparation Kit for 75 bp Single-End sequencing on Solexa platform. The sequence was directly determined by sequencing-by-synthesis technology via the TruSeq SBS kit (Illumina Inc., USA). Raw sequences were obtained from the Illumina Pipeline software bcl2fastq v2.0, which was expected to generate 30 million reads per sample. Qualified reads after filtering low-quality data were analyzed using TopHat/Cufflink. Quantification for gene expression was calculated as fragments per kilobase of transcript per million mapped reads (FPKM).
Project description:Next generation sequencing analysis was performed to identify potential target genes of lncRNA BLACAT2. Purified total RNA (3 μg) was used to deplete ribosomal RNA with Ribo-Zero rRNA Removal Kits (MRZMB126,Epicentre). Poly(A)+ RNA was purified with oligo(dT) magnetic beads and fragmented into short sequences. The library was prepared with TruSeq Stranded mRNA LT Sample Prep Kit (Cat. No.15032612,Illumina) according to manufacturer’s instructions. Each library was sequenced on an Illumina Hiseq2500 in 125PE mode (Illumina, San Diego, CA, USA). These data provided transcriptional changes in bladder cancer cell line UM-UC-3 after BLACAT2 depletion.
Project description:Purpose: We investigated the effect of the T4 MotB protein on E. coli gene expression Method: BL21(DE3) E. coli containing either pNW129 or pNW129-MotB were grown to early log phase (OD600 ~ 0.3) then induced with 0.2% arabinose for 20 minutes. Cells were then harvested and total RNA was isolated. The cDNA library was prepared using a modified RNATagSeq workflow as previously described (Shishkin, A.A. et al. 2015 Nat Methods). Optimum fragmentation of the total RNA samples in this library was determined to be 3 min at 94C in FastAP buffer (Thermo Fischer Scientific). The cDNA Library was run on a Bioanalyzer using the Agilent High Sensitivity DNA Kit to evaluate the quality of the library. The concentration of the cDNA library was determined by qPCR using the KAPA Library Quantification Kit (Kapa Biosystems, Wilmington, MA, USA) and CFX96 Real-Time PCR Detection System (Bio-Rad, Hercules, CA, USA). Sequencing was performed by the NIDDK Genomics Core facility using a MiSeq system with the single-end 50 bp Sequencing Kit (Illumina, San Diego, CA, USA). RNA-seq data was processed as previously described using E. coli B str. DE3 (NC_012971.2) as the reference genome. Differential expression between conditions was represented as a fold change, and genes with both a fold change ≥2 and adjusted p value ≤ 0.05 were considered significant. Results: RNA-seq data revealed that the expression of genes encoded in the cryptic prograge Lambda DE3 as well as an additional 29 of E. coli genes were significantly increased after motB expression. Conclusion: T4 MotB is a DNA binding protein that compacts host DNA and disrupts H-NS dependent repression.
Project description:Objective: To evaluate the characteristics of IPF lungs in fibroblasts, we performed RNA-Sequencing of fibroblasts derived from normal and IPF lungs Method: NHLF(Normal human lung fibroblast) and DHLF-IPF (DIPF; Diseased human lung fibroblasts derived from idiopathic pulmonary fibrosis) were purchased from Lonza (Walkersville, MD, USA) .Total RNA was extracted from fibroblasts (NHLF and DIPF) using an RNeasy® Mini Kit (#74106; Qiagen, Valencia, CA, USA). Preparation of a next-generation sequencing library was performed using a SMARTer® Stranded Total RNA Sample Prep Kit–Pico Input Mammalian (TaKaRa, Shiga, Japan). Sequencing was performed on an Illumina HiSeq 2500 platform in 75-base single-end mode with Illumina Casava 1.8.2 software for base calling. Sequenced reads were mapped to the human reference genome sequence (hg19) using TopHat v.2.0.13 software, in combination with Bowtie 2 v.2.2.3 and SAMtools v.0.1.19. Result: Of the 26,257 genes analyzed, for NHLF and DHLF-IPF (DIPF), FPKM was required to be greater than or equal to 0.3. Under these conditions, 764 genes were up regulated inDHLF-IPF (DIPF) and 691 genes were down regulated.