MRNA-seq study performed on MCF-7 breast cancer cells treated with a combination of vitamins A and D
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ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE221019 | GEO | 2025/07/30
REPOSITORIES: GEO
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Project description:mRNA-seq: HCT-116 colon cancer cells were grown and maintained and treated with a combination of Vitamin D2 and D3 and all trans-retinoic acid (ATRA) at 1 µM for four hours. The cells were harvested, RNA was isolated and analyzed for integrity using Agilent 4200 TapeStation and RNA Screen Tape. RNA-seq libraries were prepared using a Universal Plus mRNASeq kit, and library fragment size distribution was confirmed by electrophoresis using a 2200 TapeStation system with D1000 ScreenTape. Sequencing was carried out on NovaSeq 6000, SP flowcell, 2x50 nt reads. General quality-control metrics were obtained using FastQC and raw reads were aligned to human reference genome hg38 using STAR and BWA MEM. ENSEMBL genes were quantified using FeatureCounts, and differential expression statistics were computed using edgeR. Specific gene expression was measured and verified using qPCR. miRNA-seq: HCT-116 cells were cultured in McCoy’s 5a Medium (Gibco, Life Technology, Grand Island, NY, USA) supplemented with 10% FBS and 1% Penicillin/Streptomycin and incubated at 37°C in a humidified atmosphere of 5% CO2 and 95% air. At 80% confluence, cells were harvested by adding 0.25% trypsin/EDTA and counted by means of Trypan blue and hemocytometer. The cells were then re-suspended at appropriate concentration and plated in 96 or 6 well plates for cellular assays. Cell were treated with ATRA+D2+D3 at the IC50 concentration (1mM), then harvested and RNA isolated using Trizol
2024-12-31 | GSE160109 | GEO
Project description:Total RNA was extracted from E18.5 brains with the olfactory bulbs removed using the MirVana miRNA isolation kit (Invitrogen). RNA concentration was determined in a NanoDrop 8000 (ThermoFisher) and RNA integrity using both 2100 Bioanalyzer and 2200 TapeStation (Agilent). Libraries were prepared from 1 μg of total RNA with TruSeq RNA Sample Preparation Kit v2 (Illumina). Library size was confirmed using 2200 TapeStation and High Sensitivity D1K screen tape (Agilent) and concentration was determined by Library quantification kit (KAPA). Libraries were multiplexed five per lane and then sequenced in a HiSeq2500 (Illumina) to generate 50 million paired end 75 bp reads. The "SAMPLE_ID" sample characteristic is a sample identifier internal to Genentech. The ID of this project in Genentech's ExpressionPlot database is PRJ0013017
2018-11-14 | GSE111564 | GEO
Project description:Purpose: Identification of miRNA expression profiles of selected horse tissues. Methods: miRNA-seq analysis was performed on lung, heart and liver samples collected from 4 horses. The miRNA libraries were constructed from total RNA using NEBNext Multiplex Small RNA Library Prep Set for Illumina (New England Biolabs) according to the manufacturer protocol. The quantification of the obtained libraries was performed on a Qubit 2.0 spectrophotometer (Invitrogen, Life Technologies), while a quality control on a TapeStation 2200 instrument (D1000 ScreenTape; Agilent). 75 single-end cycle sequencing was performed on the NextSeq 500/550 platform (Illumina) with the use of NextSeq High Output Reagents v2 (Illumina). Results: The comparison of miRNA profiles between the investigated tissues revealed 185 differentially expressed microRNAs (p adj≤0.05) in the heart samples versus the liver samples, 172 DE miRNAs (p adj ≤0.05) in the lung samples in comparison to the liver samples, and 155 in the lung samples versus the heart samples. Conclusions: Obtained results show varied miRNA expression profiles characteristics for each investigated hore tissue.
2023-06-01 | GSE202502 | GEO
Project description:We generated C57BL/6 mice lacking Bmp10 and/or Bmp9 utilizing the Cre-loxP system. Briefly, Bmp9 constitutive deletion resulted from the replacement of exon 2 by a neomycin resistance cassette. Because Bmp10 deletion leads to early embryonic lethality, we used the tamoxifen-inducible Cre system to generate Bmp10-cKO mice (Rosa26-CreERT2;Bmp10lox/lox) by crossing Rosa26-CreERT2 mice with Bmp10lox/lox mice that possess loxP sites flanking exon 2. To generate double-KO (DKO) mice, we crossed these Rosa26-CreERT2;Bmp10lox/lox mice with Bmp9-KO mice. At the age of 8 weeks, mice were treated with tamoxifen (Sigma) by intraperitoneal injection once a day for 5 days at a dosage of 50 mg/kg. At the age of 5 months, Wild Type and DKO mouse lung tissue was flash frozen in liquid nitrogen and stored at -80°C. RNA extraction, RNA sample quality assessment, RNA library preparation, sequencing and raw data analysis were conducted at GENEWIZ, Inc. (South Plainfield, NJ, USA). Total RNA was extracted from frozen tissue using the Qiagen RNeasy Plus Mini kit. RNA samples were quantified using Qubit 2.0 Fluorometer (Life Technologies, Carlsbad, CA, USA) and RNA integrity was checked with Agilent TapeStation (Agilent Technologies, Palo Alto, CA, USA). rRNA depletion was performed using Ribozero rRNA Removal Kit (Illumina, San Diego, CA, USA). RNA sequencing library preparation used NEBNext Ultra RNA Library Prep Kit for Illumina by following the manufacturer’s recommendations (NEB, Ipswich, MA, USA). Briefly, enriched RNAs were fragmented for 15 minutes at 94 °C. First strand and second strand cDNA were subsequently synthesized. cDNA fragments were end repaired and adenylated at 3’ends, and universal adapter was ligated to cDNA fragments, followed by index addition and library enrichment with limited cycle PCR. Sequencing libraries were validated using the Agilent Tapestation 4200 (Agilent Technologies, Palo Alto, CA, USA), and quantified by using Qubit 2.0 Fluorometer (Invitrogen, Carlsbad, CA) as well as by quantitative PCR (Applied Biosystems, Carlsbad, CA, USA). The sequencing libraries were clustered on one lane of a flowcell. After clustering, the flowcell was loaded on the Illumina HiSeq 4000 instrument (or equivalent) according to manufacturer’s instructions. The samples were sequenced using a 2x150 Paired End (PE) configuration. Image analysis and base calling were conducted by the HiSeq Control Software (HCS). Raw sequence data (.bcl files) generated from Illumina HiSeq was converted into fastq files and de-multiplexed using Illumina's bcl2fastq 2.17 software. One mis-match was allowed for index sequence identification. After investigating the quality of the raw data, sequence reads were trimmed to remove possible adapter sequences and nucleotides with poor quality using Trimmomatic v.0.36. The trimmed reads were mapped to the the Mus musculus GRCm38 reference genome available on ENSEMBL using the STAR aligner v.2.5.2b. Gene counts were calculated from uniquely mapped reads using feature Counts from the Subread package v.1.5.2. Only unique reads that fell within exon regions were counted. The gene hit counts table was then used for downstream differential expression analysis. A differential gene expression analysis between WT and DKO groups of samples was performed using the R-package DESeq2 (Wald test).
2021-06-01 | E-MTAB-10392 | biostudies-arrayexpress
Project description:Purpose: to study the transcriptional changes that are induced by activating NR2F1 using an agonist (C26) that we developed. Methods: T-HEp3-GFP cells were pretreated with DMSO or 0.5 µM C26 for 6 days then inoculated on CAM and treated every 24 hours. After 7 days, tumors were collected and dissociated into single cells. FACS was used to isolate T-Hep3 tumor cells using GFP. RNA was extracted from fresh frozen cell pellets using Qiagen RNeasy Plus Universal mini kit following manufacturer’s instructions (Qiagen, Hilden, Germany). RNA samples received were quantified using Qubit 2.0 Fluorometer (Life Technologies, Carlsbad, CA, USA) and RNA integrity was checked using Agilent TapeStation 4200 (Agilent Technologies, Palo Alto, CA, USA). RNA sequencing libraries were prepared using the NEBNext Ultra RNA Library Prep Kit for Illumina following manufacturer’s instructions (NEB, Ipswich, MA, USA). Briefly, mRNAs were first enriched with Oligo(dT) beads. Enriched mRNAs were fragmented for 15 minutes at 94°C. First strand and second strand cDNAs were subsequently synthesized. cDNA fragments were end repaired and adenylated at 3’ ends, and universal adapters were ligated to cDNA fragments, followed by index addition and library enrichment by limited-cycle PCR. The sequencing libraries were validated on the Agilent TapeStation (Agilent Technologies, Palo Alto, CA, USA), and quantified by using Qubit 2.0 Fluorometer (Invitrogen, Carlsbad, CA) as well as by quantitative PCR (KAPA Biosystems, Wilmington, MA, USA). The sequencing libraries were clustered on 1 lane of a flowcell. After clustering, the flowcell was loaded on the Illumina HiSeq instrument (4000 or equivalent) according to manufacturer’s instructions. The samples were sequenced using a 2x150bp Paired End (PE) configuration. Image analysis and base calling were conducted by the HiSeq Control Software (HCS). Raw sequence data generated from Illumina HiSeq was converted into fastq files and de-multiplexed using Illumina's bcl2fastq 2.17 software. One mismatch was allowed for index sequence identification. Three replicate samples per group were used . R (v.4.0.3) was used to perform bioinformatics analysis. Quality control was performed using FastQC (v0.11.8). Trim Galore! (version 0.6.5) was used to trim the adapter sequences with a quality threshold of 20. The human genome reference used was GRCh38.p13 and GENCODE release 36. Alignment was performed using STAR aligner (v2.7.5b). Gene level read counts were obtained by using Salmon (v1.2.1) for all libraries . All samples passed the quality control requirements with > 60% of reads uniquely mapping (>20M uniquely mapped reads for each library) using STAR. Differential expression analysis was performed using the gene level read counts and the DESeq2 (v1.28.1) R package. Heatmaps show the z-scores of gene-level read counts and were generated using heatmaply (v1.1.0). Genes with adjusted p-value less than 0.05 were considered differentially expressed.
2021-04-02 | GSE171353 | GEO
Project description:Monocyte-derived macrophages were stimulated with 25 ng/ml of the indicated IFNs or infected with HIV-1-BaL-HSA. Total RNA was isolated 18 h following stimulation/infection using RNeasy minikit (Qiagen). Intact poly(A) RNA was purified from total RNA samples (100 to 500 ng) with oligo(dT) magnetic beads, and stranded mRNA sequencing libraries were prepared as described using the Illumina TruSeq Stranded mRNA Library Preparation kit (catalog no. RS-122-2101 and RS-122-2102). Purified libraries were qualified on an Agilent Technologies 2200 TapeStation using a D1000 ScreenTape assay (catalog no. 5067-5582 and 5067-5583). The molarity of adapter-modified molecules was defined by quantitative PCR using the Kapa Biosystems Kapa Library Quant kit (catalog no. KK4824). Individual libraries were normalized to 10 nM, and equal volumes were pooled in preparation for Illumina sequence analysis. Sequencing libraries (25 pM) were chemically denatured and applied to an Illumina HiSeq v4 single-read flow cell using an Illumina cBot system. Hybridized molecules were clonally amplified and annealed to sequencing primers with reagents from an Illumina HiSeq SR cluster kit v4-cBot (catalog no. GD-401-4001). Following transfer of the flow cell to an Illumina HiSeq 2500 instrument (HCSv2.2.38 and RTA v1.18.61), a 50-cycle single-read sequence run was performed using HiSeq SBS kit v4 sequencing reagents (catalog no. FC-401-4002).
2020-10-02 | GSE158434 | GEO
Project description:Intestinal explants from 5-weeks old piglets were exposed 4h to control or 10 µM deoxynivalenol-contaminated cell culture medium. Explants were nitrogen frozen. RNA were extracted, quantified and their quality was checked with Tapestation 4200 device. RNA samples were used to perform a microarray experiment
2021-06-30 | GSE165968 | GEO
Project description:The purpose of the experiment was to determine whole transcriptome changes after spinal cord injury (SCI) to understand how photobiomodulation promotes neuroprotection and functional recovery. SCI was performed at the thoracic (T) level T8, by crushing the dorsal columns using a calibrated watchmaker's forceps. Animals were then treated with photobiomodulation (PBM; 660nm wavelength) or sham control light, within 15 minutes of the injury. PBM or sham-treatment was treatment directed at the lesion site for 1 minute duration every 24hr for the first 3 days. Animals were then killed, tissues harvested and the RNA was extracted using RNEasy lipid tissue mini kit (Qiagen) according to the manufacture's instructions. Whole genome sequencing was outsourced to Qiagen RNA Sequencing Services (Germany).Library preparation was performed using the QIAseq Stranded Total RNA Library Kit with QIAseq FastSelect rRNA and globin depletion. QIAseq FastSelect rRNA HMR was used to reduce the amount of unwanted RNA species. After first and second strand synthesis, the cDNA was end-repaired and 3’ adenylated. Sequencing adapters were ligated to the overhangs. Adapted molecules were enriched using 16 cycles of PCR and purified by a bead-based cleanup. Library preparation was quality controlled using capillary electrophoresis (High Sensitivity Tape D1000) and high-quality libraries were pooled based on equimolar concentrations. The library pool(s) were quantified using qPCR and optimal concentration of the library pool used to generate the clusters on the surface of a flowcell before sequencing on a NovaSeq (Illumina Inc., Madison, USA) instrument (2x75, 2x10) according to the manufacturer instructions (Illumina Inc.). Raw data was de-multiplexed and FASTQ files for each sample were generated using the bcl2fastq software v2.20.0.422 (Illumina inc.).
2024-11-21 | E-MTAB-14593 | biostudies-arrayexpress
Project description:To obtain a preliminary picture at the transcriptional level of the set of genes linked to the regulatory activity of the small RNA GssA of P. aeruginosa PA14, we analyzed the global transcription profile of PA14deltagssA strain in comparison with PA14 by an RNA-seq approach, extracting total RNA at OD600 of 2, corresponding to early stationary phase, when the two strains were grown in Brain Heart Infusion (BHI) rich medium, a condition in which, at the time of its identification, a relevant expression of GssA was noted. For each strain, three independent biological replicates were performed. The concentration and the purity of the RNA starting material were determined spectrophotometrically while RNA Integrity Number (RIN) was measured on an Agilent TapeStation 4200. An additional DNase step was included to cleave DNA that could interfere with rRNA removal and negatively affect library preparation and sequencing. RNA libraries were generated starting from 3 μg of total RNA. rRNA was depleted with Illumina Ribo-Zero rRNA Removal Kit (Gram-Negative Bacteria) and libraries were generated according to Illumina TruSeq® Stranded mRNA Library Prep protocol. RNA libraries were accurately quantified with a fluorometric method: QUBIT dsDNA HS assay kit and the size and purity were verified on an Agilent TapeStation 4200. RNA libraries were then sequenced on an Illumina HiSeq with paired-end reads 150 bp long. For RNA-Seq data analysis, P. aeruginosa UCBPP-PA14 (NCBI: NC_008463.1) genome and annotation (GTF) were downloaded from the Pseudomonas Genome Database. Alignment of paired Fastq RNA-Seq reads to P. aeruginosa UCBPP-PA14 genome was performed using the STAR aligner (v2.7.0) with the quantMode TranscriptomeSAM GeneCounts option turned on. SAM files were converted to BAM, sorted, and indexed using SAMtools. Differential gene expression analysis was performed using DESeq2 using a design = ~ condition model and dedicated R scripts. The false discovery rate was controlled using the Benjamini-Hochberg procedure, with an adjusted p-value (FDR) ≤ 0.1. P. aeruginosa gene IDs were annotated with a gene description field using a custom tool (IdToGeneNamesScript) using the P. aeruginosa UCBPP-PA14 GTF file as annotation input.
2022-11-30 | E-MTAB-12073 | biostudies-arrayexpress
Project description:The goal of this study is to identify novel target genes of DVL3 in two breast cancer cell lines Methods:Total RNA was isolated using the Aurum™ Total RNA Mini Kit (Bio Rad) and library preparation and sequencing were performed at Center for Biotechnology & Genomics of Texas Tech University. RNA quality was determined using RNA Screen Tape (Agilent). Ribosomal RNA depletion was achieved using NEB Next rRNA Depletion Kit (Human/Mouse/Rat) (NEB # E6310X). RNA fragmentation, double stranded cDNA and adaptor ligation was generated using NEBNext Ultra II Directional RNA Library Prep according to the manufacturer’s protocol (NEB # E7760L). PCR enriched libraries were quantified by Qubit and equimolar indexed libraries (different samples had different indexes for multiplexing) were pooled. Pooled libraries were quantitatively checked using the Agilent Tapestation 2200 and quantified using Qubit. The libraries were then diluted to 200 pM and spiked with 2% phiX libraries (Illumina control). The transcriptome sequencing was performed on the barcoded stranded RNA-Seq libraries using Illumina NovaSeq 6000 SP flow cell, paired-end reads (2 × 50 bp).
2021-04-16 | GSE165773 | GEO