Analysis of CAL165 cells transcriptome using RNASeq
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ABSTRACT: Total RNA extracted from the head and neck cell line CAL165 were purified using miRNeasy minikit (Qiagen). RNA libraries were then generated with the NEB next small library prep set for SOLID (New England Biolabs) and sequenced on the Applied Biosystems SOLiD 5500 wildfire system following the manufacturer's instructions.
Project description:Total RNA extracted from biopsies of healthy skin, papilloma, SCC Tumor and of 2 cell lines isolated from these tumors were purified using miRNeasy minikit (Qiagen). RNA libraries were then generated with the NEB next small library prep set for SOLID (New England Biolabs) and sequenced on the Applied Biosystems SOLiD 5500 wildfire system following the manufacturer's instructions.
Project description:The molecular mechanisms governing heart development provide an important framework to understand congenital heart disease. The embryonic vertebrate heart tube develops an atrioventricular canal that divides the atrial and ventricular chambers, forms atrioventricular conduction tissue and organizes valve development. To better understand the molecular mechanism underlying atrioventricular canal versus chamber myocardium expression, a double-reporter transgenic mouse line was generated in which the expression of EGFP (green fluorescent protein) and Katushka (red fluorescent protein) are selectively expressed in the atrioventricular canal and in the chamber myocardium, respectively. We assessed the genome-wide H3K27ac pattern in isolated embryonic AV canal and in chamber cardiomyocytes, respectively. EGFP and Katushka-positive cells were purified by FACS and fixed. ChIP was performed with the truemicro ChIP (Diagenode) according to the manufacturer’s protocol using the H3K27ac antibody (abcam ab4729). Chipped DNA were subjected to library preparation using the 5500 Series SOLiD™ Systems ample preparation kit (Applied Biosystems) according to manufactures recommendations and sequenced using the 5500 wildfire system (SOLiD).
Project description:Mouse BM extracellular vesicles were isolated from Ocn-GFP Topaz mice using the Exoeasy and miRneasy (Qiagen). Small RNA libraries were constructed using NEBNext Multiplex Small RNA Library Prep Set for Illumina (New England biolabs). Libraries were sequenced using Hiseq2500 instrument
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:Total RNA from cells in nasal turbinate was extracted with miRNeasy micro kit (Qiagen, 217084) according to the manufacturer’s instructions. ERCC RNA Spike-In Mix kit (ThermoFisher Scientific, 4456740) was added to normalized total RNA prior to library preparation following manufacturer’s protocol. The RNA sequencing libraries were prepared using the NEBNext Ultra II RNA Library Prep Kit for Illumina according to manufacturer’s instructions (New England Biolabs, Ipswich, MA, USA). The samples were sequenced by the Illumina HiSeq instrument according to manufacturer’s instructions using a 2x150bp Paired End (PE) configuration.
Project description:In a cross-site study we evaluated the performance of ribosomal RNA removal kits from Illumina, Takara/Clontech, Kapa Biosystems, Lexogen, New England Biolabs and Qiagen on intact and degraded RNA samples. We found that all of the kits were capable of performing significant ribosomal depletion, though there were differences in their ease of use. All kits were able to remove ribosomal RNA to below 20% with intact RNA and identify ~14,000 protein coding genes from the Universal Human Reference RNA sample at >1FPKM. Analysis of differentially detected genes among kits suggested that transcript length may be a key factor in library production efficiency. These results provide a roadmap for labs on the strengths of each of these methods and how best to utilize them.
Project description:For the AcceSssIble assay, nuclei preparation and M.SssI methyltransferase (New England BioLabs) treatment were performed. The subsequent Infinium DNA methylation assay was performed at the University of Southern California Molecular Genomics Core Facility according to the manufacturer’s specifications (Illumina).
Project description:Maternal diabetes is a teratogen that can lead to neural tube closure defects in the offspring. We therefore sought to compare gene expression profiles at the site of neural tube closure between stage-matched embryos from normal dams, and embryos from diabetic dams. Neurulation-stage mouse embryos at 8.5 days of gestation were used to prepare neural tissue at the anterior aspect of neural tube closure site 1. Tissue was procured from the open neural tube immediately anterior of the closure site, and from the closed neural tube immediately posterior to the closure site by laser microdissection. For each sample, 10 sections were pooled, total RNA was extracted, and 7 ng of total RNA were used for expression profiling by Tag sequencing using an Applied Biosystems SolidSAGE kit for library construction, and an AB SOLiD 5500 XL instrument for sequencing. Sequence reads were mapped to RefSeq RNA, and count data per gene were obtained using a modified version of the Applied Biosystems SOLiD™ SAGE™ Analysis Software.
Project description:Maternal diabetes is a teratogen that can lead to neural tube closure defects in the offspring. In neurulation-stage embryos from diabetic dams, we detected abnormal tissue protruding from the open neural tube. To determine the origin of such protrusions, we compared gene expression profiles between open neural plate with normal morphology, and protrusion tissue. Neurulation-stage mouse embryos at 8.5 days of gestation were used to prepare open neural tube at the anterior aspect of neural tube closure site 1 by laser capture microdissection. For each sample, 10 sections were pooled, total RNA was extracted, and 7 ng of total RNA were used for expression profiling by Tag sequencing using an Applied Biosystems SolidSAGE kit for library construction, and an AB SOLiD 5500 XL instrument for sequencing. Protrusion tissue was prepared from whole embryos by microdissection, and 12ng of total RNA per sample was used for Tag sequencing. Sequence reads were mapped to RefSeq RNA, and count data per gene were obtained using a modified version of the Applied Biosystems SOLiD™ SAGE™ Analysis Software.
Project description:RNA was purified from serum of osteoporotic and healthy postmenopausal mexican women using miRNeasy Serum/Plasma kit (QIAGEN). For microRNA expression analysis we used the Human MicroRNA A+B Cards Set v3.0 TaqMan Low Density Array platform (Applied Biosystems). Analysis was performed in the Expression Suite v.1.1.3 software (Applied Biosystems).