Project description:Total RNA, greater than 100ng, from cultured cells was isolated in TRIzol L and purified using Qiagen RNeasy Mini Kit per manufacturer’s protocols. Agilent Technologies 2100 Bioanalyzer was used to assess the RNA quality. RNA libraries were prepared and multiplexed using Illumina TruSeq RNA Library Preparation Kit v2 (non-stranded and poly-A selection) and 10 nM of cDNA was used as the input for high-throughput sequencing via Illumina’s HiSeq 2500 platform, producing 50 bp paired end reads.
Project description:To analyze Gq-receptor hypertrophic transcriptional profiles, 10 to 11 week old FVB/NJ mice were dosed at 30 mg/kg/day for phenylephrine and 0.5 mg/kg/day for angiotensin II using implanted micro-osmotic pumps (Alzet, model 1002) for 72 hours. Total RNA was harvested from isolated myocytes using RNeasy Fibrous Tissue Mini Kit (Qiagen), following manufacturer’s instructions. Using total RNA as input, rRNA was removed via oligo-dT purification to enrich for mRNA, followed by random-primed reverse-transcription, second-strand cDNA synthesis, ligation of indexed adaptors for Illumina sequencing, and subsequent library creation from the resulting double-stranded DNA. 125 base pair paired end stranded RNA Sequencing was performed by the University of Minnesota Genomics Core using the Illumina HiSeq 2500 resulting in 10-20 million reads per sample.
Project description:In this study, we used RNA-Seq to understand the mechanisms of Cd toxicity, cellular detoxification and protection pathways in response to Cd in rice roots. To gain additional insight into the rice transcriptomic response to environmental Cd stress, 15-day-old rice seedlings were treated with 10 or 100 μM solutions of Cd2+, or without Cd (control), for 24 h, at which point root samples were harvested and labeled as Cd+, Cd++, and control, respectively. These samples were used for 101 bp paired-end (PE) deep sequencing on an Illumina HiSeq 2500 platform.
Project description:To address how Csf3r and RUNX1 mutations in combination with CSF3 administration affect hematopoiesis in vivo, we performed serial transplantation experiments. Lineage-negative Csfr-d715 BM cell cells were lentivirally transduced with the patient specific RUNX1-D171N (RHD) mutant or an empty vector control. Primary recipients showed a pre-leukemic condition characterised by myeloblasts in the peripheral blood, but not overt AML. Upon serial transplantation, one of the Csfr-d715/RUNX1-D171N mutant mice, treated with CSF3, could repopulate secondary and tertiary recipients. Whole exome sequencing on these mice were performed to investigate whether these mice acquired an additional mutation. Enzymatically fragmented genomic DNA was used to construct sample libraries following the SeqCap EZ HyperPlusCap workflow User’s Guide version 1.0 (Roche). Unique, dual index adapters (Integrated DNA technologies) were used for ligation. After ligation of adapters and an amplification step, exome target sequences were captured using in-solution oligonucleotide baits (SeqCap EZ Developer Library mm9_exome_L2R_D02). Amplified captured sample libraries were paired-end sequenced on the HiSeq 2500 platform (Illumina).
Project description:Our goal was to identify early genetic changes in the development of autoimmune dysfunction. WT and IL-2-KO CD8 T cells were sorted from the lymph node and spleen of 12-day old mice. Total RNA was isolated by Expression Analysis Inc. using Illumina TrueSeq Stranded Total RNA Sample Preparation Kit. Eight samples were sequenced (four biological replicates of IL-2-KO and WT/HET mice), producing 2X50 paired-end reads using the Illumine HiSeq 2500 platform. Raw reads were provided by Expression Analysis. We identified several genetic signatures within the bulk data including a cytolyic pattern and a novel gene expression pattern indicating a helper-like function.
Project description:Sheep total RNA was extracted from the Ileo-caecal valve lymph node (ICLN) of sheep with multibacillary paratuberculosis compared to ICLN of uninfected controls. Sequencing libaries were prepared from RNA using the Illumina TruSeq RNA Sample Preparation Kit v2. Sequencing with 101 base paired end reads was performed on an Illumina HiSeq 2500 at Edinburgh Genomics.
Project description:In this study, RNA-seq was used to compare the transcriptomes of Listeria monocytogenes 10403S and H7858 and corresponding ΔsigB mutants under exposure to pH 5.5 with or without bile. RNA-seq was performed on all strains and conditions in four independent biological replicates. Indexed and purified cDNA libraries (12 libraries per strains) were loaded together onto an independent flow cell without any other samples; sequencing was carried out by running Hiseq 2500 (single-end, 100-bp per read). Reads alignment was carried out using the Burrows-Wheeler Aligner (BWA). Differential expression of genes in different strains and conditions was statistically assessed using the BaySeq method.
Project description:CD3+ T cells were isolated by FACS from primary tumour tissues of two triple-negative breast cancer patients. Sorted cells were submitted to a 10X Genomics Chromium System for single cell capture. cDNA synthesis and library preparation were done according to the protocol supplied by the manufacturer. Libraries were sequenced on an Illumina HiSeq 2500 High Output Mode using V4 clustering and sequencing chemistry to achieve 100 bp paired-end reads.
Project description:Samples of human total RNA from different brain regions at embryonic and adult life stages, along with samples from testes (used as a control for germline piRNAs), were obtained from BioChain Institute (CA, USA) and TaKaRa Bio Inc. (CA, USA). Indexed libraries were prepared using 1 µg of total RNA and the TruSeq Small RNA Sample Prep Kit (Illumina, CA, USA), following the manufacturer’s instructions. Each library was sequenced at a concentration of 3pM for 50 cycles on the HiSeq 2500 platform (Illumina, CA, USA). The dataset presented encompasses small non-coding RNA expression profiles across various human brain regions and may be used for further studies of small RNA-related molecular mechanisms of brain function and disease.