Next Generation Sequencing Facilitates Quantitative Analysis of Wild Type and Ptprz1-/- lung microvascular endothelial cell (LMVEC) Transcriptomes
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ABSTRACT: Purpose: To compare NGS-derived transcriptome profiling (RNA-seq) between Wild Type and Ptprz1-/- LMVEC and identify transcripts that may be linked to the phenotypic differences observed. Methods: Total RNA from freshly cultured LMVEC was extracted using the NucleoSpin RNA Plus kit from Macherey-Nagel and the concentration and purity was assessed by Nanodrop and gel agarose 1% in TBE 0.5x. RNA from 3 independent fresh cell isolations from the Ptprz1+/+ and the Ptprz1-/- LMVEC was extracted and sent for RNAseq analysis. Novogene Co., Ltd performed the mRNA isolation, cDNA library synthesis and sequencing, and the bioinformatics analysis. Results: Pairwise comparison of gene expression of Ptprz1-/- RNA (3 samples) against Ptprz1+/+ RNA (3 samples) identified several transcripts as differentially expressed. Twenty-six genes were significantly changed (adj. p value <0.05), with 9 being upregulated and 17 downregulated. These genes are linked to angiogenesis and heart development and function. Conclusions: Our study represents the first transcriptomic analysis of Ptprz1-/- endothelial cells compared to the corresponding wild-type cells, generated by RNA-seq technology.
Project description:To assess changes in immune gene expression following HIV-env vaccination through 3 delivery systems, rhesus macaques were vaccinated with SOSIP trimer with adjuvants using either oral, intraepithelial delivery through a syringe (IEP), a needle free pressurized oral delivery (NF), or an intramuscular injection in the right deltoid (IM). Buccal mucosal samples were collected by cytobrush and stored in Nucleospin lysis buffer RA1 with 1% β-mercaptoethanol and RNA was extracted using Macherey Nagel nucleospin columns. RNA from both timepoints was then used for gene expression analysis using the Nanostring nCounter platform.
Project description:To assess changes in immune gene expression following BCG vaccination in infants, 6 infant rhesus macaques were vaccinated with BCG at 2 weeks of age. Whole blood was collected at the time of BCG vaccination, and at 3 weeks post-BCG, in EDTA coated tubes. Peripheral blood mononuclear cells were collected by ficoll and RNA was extracted using Macherey Nagel nucleospin columns. RNA from both timepoints was then used for gene expression analysis using the Nanostring nCounter platform.
Project description:To assess changes in immune gene expression following HIV-env vaccination through 3 delivery systems, rhesus macaques were vaccinated with SOSIP trimer with adjuvants using either oral, intraepithelial delivery through a syringe (IEP), a needle free pressurized oral delivery (NF), or an intramuscular injection in the right deltoid (IM). Submandibular lymph nodes were biopsied and stored in RPMI supplemented with 10% fetal bovine serum. RNA was extracted using Macherey Nagel nucleospin columns. RNA from both timepoints was then used for gene expression analysis using the Nanostring nCounter platform.
Project description:Total RNA from shMLL1 Ls174T cells induced with 300ng/ml doxycycline for 3 days and non-induced parental cells was isolated with the NucleoSpin RNA isolation kit (Macherey-Nagel) and sequenced on a NextSeq 500 (Illumina).
Project description:A panel of twelve breast cancer cell lines (ATCC) were exposed to 0, 1 or 10 nM 17β-estradiol (E2) for 16 hours. Cells were maintained in DMEM/F12 1:1 (DF) supplemented with 10% or 20% fetal calf serum (FCS) or 20% FCS and 0.01 mg/ml insulin. Two days before experiments, cells were seeded in 60 mm plates in DF with charcoal stripped phenol red-free serum supplemented with non-essential amino-acids and pen/strep, at a density such that 75%-90% confluence was obtained by the time of harvesting. Medium was refreshed 24 hours before exposure to 1 nM E2, 10 nM E2 or DMSO (control) for 16h. Subsequently cells were harvested and RNA was extracted using the Nucleospin RNA isolation kit (Macherey-Nagel).
Project description:affy_ath_2012_04 - affy_ath_2012_04 - Epigenetic informations include histone post-translational modifications such as acetylation and methylation. The jumoni proteins can remove the methyl group from lysine or arginine residues of histones. The aim of this project is to compare gene expression levels between mutants of two jumonji genes and wild type at seedling stage (12 d old).-- Seeds were sterilized in bleach, washed with ethanol, dried, dispersed on Murashige and Skoog (MS) 0.5X containing 2% sucrose and kept 5 days at 4°C. - After 12 days of growth under a cool white fluorescent light (120 μmol m-2 s-1) and long day conditions (16h of light) in a growth chamber, the plants are harvested and stored at -80°C. -The RNA are extracted with the RNA Plant NucleoSpin (Machery-Nagel), quantified with Nanodrop and visualised on agarose gel. The samples are stored at -80°C.
Project description:Interleukin 2 (IL-2) promotes proliferation and differentiation of CD8+ T cells in vitro and in vivo. To define gene expression regulated by IL-2, we purified naive CD8+ T cells, activated them for 2 days followed by treatment with recombinant IL-2 or with neutralizing antibody against IL-2 and compared gene expression between the two treatments. Total RNA was extracted using the NucleoSpin RNA XS kit (Macherey-Nagel), amplified using a PicoSL RNA amplification kit (Nugen) and biotinylated with Encore biotin module (Nugen). Labeled RNA was hybridized to Mouse Gene 1.0ST microarrays (Affymetrix) according to the manufacturer’s instruction.
Project description:5000-10000 HSCs (KL CD150+ CD48- CD34-) were sorted into lysis buffer from the pools of naive or 1-month infected WT or Dnmt3a-/- mice (n=10-12 per group). RNA was isolated with the NucleoSpin ® RNA Plus XS kit (Macherey Nagel). RNA-seq libraries were prepared by using SMARTer® Stranded Total RNA-Seq Kit v2 - Pico Input Mammalian (Takara Bio Usa). Illumina NovaSeq SP was used for sequencing with a paired-end sequencing length of 10bp. FASTQ files were preprocessed using HTS stream (https://github.com/ibest/HTStream) and the clean FASTQ file were aligned using STAR. Differential expression (DE) analysis of gene expression was performed using Limma-Voom. False discovery rate (FDR)<0.05 was considered statistically significant. We performed gene ontology analysis for differentially expressed genes with q value <0.05.
Project description:Abstract: Wallerian degeneration (WD) is a process of autonomous distal degeneration of axons upon injury. Macrophages (MP) of the peripheral nervous system (PNS) are main cellular agent controlling this process. Some evidences suggest that resident PNS-MP along with MP of hematogenous origin may be involved but whether these two subsets exert distinct functions is unknown. Combining MP-designed fluorescent reporters mice, and coherent anti-stoke raman scattering (CARS) imaging of the sciatic nerve, we deciphered the spatio-temporal choreography of resident and recently recruited MP after injury and unveiled distinct functions of these subsets with recruited MP responsible of efficient myelin stripping and clearance while resident MP were involved in axonal regrowth. This work provides clues to tackle selectively cellular processes involved in neurodegenerative diseases. Methods: (relevant for this GEO dataset): RNAseq of sciatic nerve macrophages from mice after CCI: Sorted cells were lysed in 100µl of RA1/TCEP buffer (NucleoSpin RNA XS, Macherey-Nagel), snap frozen in liquid nitrogen and stored at -80C until RNA extraction. All samples were processed in parallel and RNA extraction (without Carrier RNA but with on-column DNase treatment) was performed according to manufacturer's instructions (NucleoSpin RNA XS, Macherey-Nagel). RNA was eluted in RNAse-free water. Preparation of cDNA libraries for RNAseq was done using the SmartSeq method according to manufacturer's instructions (SMART-Seq v4 Ultra Low Input RNA Kit for Sequencing, Clontech/TaKaRa). Due to the low number of cells, the total amount of eluted RNA was used as starting material for reverse transcription, followed by 18 cycles of pre-amplification. 1ng of cDNA were used for RNAseq sequencing library preparation, according to manufacturer's instructions (Nextera XT DNA Library Preparation, Illumina). Final samples pooled library prep were sequenced on a Nextseq 500 ILLUMINA with MidOutPut cartridge (2x130Millions of 75 bases reads) with 2 runs (4plex and 5plex), corresponding to 2x30Millions of (paired-end) reads per sample after demultiplexing.
Project description:affy_petaldvt_lyon_rose. The objective is to identify genes involved in petal development in rose. We aim at identifying genes whose expression correlates with flower opening and scent emission. In this study, we used a microarray approach to compare the transcriptome of a scented rose flower (PF) versus non-scented rose flower (RF). Samples (petal tissues) were collected at the same time early in the afternoon. Total RNA was extracted using the Plant RNA kit (Macherey Nagel), and then used to hybridize Rosa-Affymetrix microarrays. Keywords: scented vs non-scented flowers