RNA-seq in wild-type Saccharomyces cerevisiae S288c cells to determine the effect of sodium crotonate treatment on the transcriptome
ABSTRACT: RNA-seq in wild-type yeast cells to determine the effect of crotonate treatment on the transcriptome. Three independent yeast colonies were used for inoculation. Overnight cultures were diluted in fresh medium with or without 10 mM sodium crotonate in the presence of 0.8 M sorbitol. After 3.5 hours cells were in early log-phase (1 × 10^7 cells/ml) and harvested by centrifugation. Total RNA was isolated using the RNeasyMini kit and treated with the RNase-Free DNase Set to remove any contaminating genomic DNA. Library preparations were performed with the TruSeq Stranded mRNA Library kit (Illumina). Three independent biological replicates for each condition were subjected to RNA-seq analysis using an Illumina NextSeq500 with a 75 bp single read flowcell.
Project description:Genomic DNA from 74 wild type Col x Ler F2 individuals was extracted using CTAB and used to generate sequencing libraries as described (Rowan et al; Yelina et al), with the following modifications. DNA was extracted from 3 rosette leaves of 5 week old plants and 150 ng of DNA used as input for each library. DNA shearing was carried out for 20 minutes at 37C with 0.4U of DNA Shearase (Zymo research). The barcoded adapters used for library construction are listed in Rowan et al. Each set of 96 libraries was sequenced on one lane of an Illumina NextSeq500 instrument (300-cycle Mid Output run). Sequencing data were analyzed to identify crossovers as previously reported, using the TIGER pipeline (Rowan et al; Yelina et al).
Project description:The experiment was designed to obtain a broader unbiased view of the changes in islet macrophages following low dose STZ challenge. Mice were purchased from Jackson Laboratory (Bar Harbor, ME). 16-20-week-old C57BL/6J males were given 30 mg/kg STZ or acetate buffer (control) i.p. (intraperitoneal injection) for 5 consecutive days. Following the first STZ or buffer injection mice were sacrificed on day 14 and islets were isolated by collagenase digestion. Freshly isolated islets were dispersed in 0.02% Trypsin-EDTA for 3 minutes followed by up to 1 minute of pipetting under a stereomicroscope to obtain a single cell solution. Islet media was added to stop the reaction. Islets from 10 mice were pooled per sample (N). Dispersed islets were washed with FACS buffer (1% heat inactivated FBS, 1 mM EDTA, 11 mM glucose in PBS). Cells were kept on ice and pre-incubated with Fc Block (1:100) for 5 minutes, followed by 30 min incubation with CD45-eFluor 450 (1:250; clone 30-F11), Ly-6C-APC (1:1,200; clone HK1.4), CD11b-PE (1:1,200; clone M1/700, F4/80-FITC (1:150; clone BM8), CD11c-PECy7 (1:150; clone N418), and the viability dye 7AAD (1:2,000). Unstained, single stains, and fluorescence minus one controls were used for setting gates and compensation. Viable, single CD45+Ly6c-Cd11b+Cd11c+F4/80+ cells were sorted using a BD FACS Aria IIu directly into lysis buffer, and the RNeasy Plus Micro Kit from Qiagen was used to isolate total RNA. Total RNA quality control quantification was performed using an Agilent 2100 Bioanalyzer. All RNA samples had an RNA integrity number (RIN) ≥9.1. The NeoPrep Library Prep System from Ilumina was used for library preparation followed by sequencing using standard Illumina methods and Ilumina NextSeq500.
Project description:The experiment was performed with the intention of collecting transcriptomic data from isolated cell types (spore, stalk and vegetative cells) in Dictyostelium lacteum as well as from cysts in Polyshpondlyium pallidum. By combining these data with similar cell-type specific RNA-Seq data from other organisms, and by examining the expression patterns of transcription factor genes, we tried to characterize how gene regulation for cell differentiation evolved in Dictyostelia. Specifically ,we dissociated and collected spore and stalk from the fruiting bodies of D. lacteum at 24 hours of development. We also collected exponentially growing vegetative cells of D. lacteum. For collecting P. pallidum cyst samples, cells were induced to encyst with sorbitol, and samples were collected at 0, 8, 16, and 24 h of incubation. RNA was extracted using the RNeasy kit (QIAGEN), and cDNA libraries were made using the Illumina TruSeq kit. The Illumina sequencing platforms (NextSeq500 for D. lacteum samples, and Hi-seq 2000 for P. pallidum samples) were used for sequencing.
Project description:We performed RNA-seq experiments on two replicate samples from each HNRNPC knockdown (KD1 and KD2) As well as from control HeLa cells. Library preparation was preformed according to mRNA Sequencing Sample Preparation Guide (Illumina, Part # 1004898 REV. D). Reagents were taken from the Illumina sample preparation kit (Illumina, CAT # RS-930-1001). Knockdown and control samples were sequenced together in one flowcell on one and two lanes, respectively.
Project description:The goal of this study is to investigate the differential transcripted genes affected by CRISPR induced endoglin knockout in PASMC cells. Total RNA was purified from NTC or ENG-/- PASMC cells using RNeasy Mini Kit (Qiagen, Hilden, Germany). RNA quality and concentration were assessed with Agilent Tapestation 200 (Agilent Technologies) and Qubit 2.0 (ThermoFisher Scientific). ~250-500 ng RNA were used for library construction. The NGS libraries were constructed using the KAPA Stranded mRNA-Seq Kits (KapaBioSystems). mRNA was captured using magnetic oligo-dT beads and 1st strand cDNA was synthesized using random priming. In order to preserve strand-specificity, 2nd strand synthesis, which converts the cDNA:RNA hybrid to double-stranded cDNA (dscDNA), was marked by dUTP incorporation. cDNA framents were A-tailed by adding dAMP to the 3'-ends of the dscDNA library fragments. dsDNA Illumina TruSeq "forked” adapters 3'-dTMP overhangs were then ligated to A-tailed library insert fragments. Each of the six libraries were ligated with a unique Truseq 6bp barcode. Library fragments were amplified using the KAPA HiFi HotStart polymerase. The strand marked with dUTP was not amplified, allowing strand-specific sequencing. Fragment length and library quality was assessed on a 2100 Bioanalyzer using the High Sensitivity DNA Kit (Agilent Technologies). Libraries were diluted to 10nM and pooled at equimolar ratios. The pool was then diluted to 2nM and denatured in NaOH following Illumina recommendations. 10pM of denatured library pool was loaded in one HiSeq lane and flowcell was clustered on the Illumina C-bot. 5% PhiX control was spiked-in. The flowcell was sequenced on a HiSeq 2500 V4 chemistry with 50bp Single read protocol. Data was demultiplexed and Fastq files were generated using BcptoFastq 1.8.4 script provided by Illumina.
Project description:Comparison of TopHat alignments and assessment of spurious splice junctions for 32nt and 76nt read lengths. Total RNA from 2-week-old Arabidopsis thaliana (ecotype Columbia) seedlings grown on MS plates was isolated using RNeasy Plant Mini Kit from Qiagen. To remove any contaminating DNA, RNA was treated with DNAse. Isolation of poly (A) mRNA and preparation of cDNA library were carried out using the Illumina TrueSeq RNA kit. Sequencing (72 cycle) was done on Illumina Genome Analyzer II. 2 replicates
Project description:Genetic variants in TCF7L2 are the most highly associated variants with type 2 diabetes.We performed RNA_seq experiments on Control and Knockdown samples for 4 passages each. Library preparation was performed according to mRNA Sequening Sample Preparation Guide (illumina). Reagents were taken from the Illumina sample preparation kit v.2. Knockdown and control samples were sequenced together in one flowcell, 6 samples per lane. The aim of this study is to identify the downstream target genes that might the involvement of TCF7L2 in the development of type 2 diabetes. No technical replicate, 4 biological replicates.
Project description:The goal of this study is to examine the effect of EIF5A knockdown on the MCF-7 transcriptome. Overall design: MCF-7 total RNA profiles upon siRNA-mediated knockdown of EIF5A or using a control siRNA (siCTRL) were generated by deep sequencing, in triplicate, using the Illumina TruSeq kit (RPHMR12126). Library products were sequenced on the Illumina NextSeq500 platform.
Project description:Hfq is a transcriptional and translational pleiotropic regulator in several bacteria. RNA-Seq, Ribo-Seq and Proteomic analyses were carried out in the wild-type and a hfq deletion strain of Pseudomonas fluorescens SBW25 with the intention to separate the influence of Hfq on the transcript stability and translation. This submission relates to the RNA-Seq data only. RNA was extracted from two replicate cultures each of SBW25-WT and SBW25-Δhfq strains and, after removal of ribosomal RNA, subjected to RNA-Seq in an Illumina NextSeq500 machine. The resulting sequence data was analysed by mapping to the reference sequence of Pseudomonas fluorescens SBW25 as available in the Genbank accession NC_012660.
Project description:m6A-seq of undifferentiated and differentiated mouse embryonic stem cell m6A-mRNA library for undifferentiated and differentiated mouse embryonic stem cell each having one biological replicate were generated using HiSeq2000 v3 flowcell (Illumina) and sequenced for 100 bases with separate 7 base indexing read in a single lane.