Project description:Comparison of transcriptome in adult control F344 kidneys versus Hsd11b2 knockout kidneys (model of syndrome of apparent mineralocorticoid excess; SAME)
Project description:SMARCD1, an auxiliary subunit of the SWI/SNF complex is essential for maintaining embryonic stem cell pluripotency and metabolic gene regulation. Here, we have tried to address the role of SMARCD1 in myeloid differentiation and leukemia. To address this, we transduced U937 cells with lentiviral particles expressing shcontrol and shSMARCD1. Next we subjected these cells to 30 nM vitamin D3 induction for 48 h. RNA-sequencing was conducted for shcontrol and shSMARCD1 in uninduced and vitamin D3 induced U937 cells. High quality RNA with RIN values above 9, was used to generate library using NEBNext Ultra II Directional RNA Library Prep Kit for Illumina (NEB #E7760) following manufacturer’s instructions. Finally barcoded libraries were pooled, and a final concentration of 1.2 pM was loaded using NextSeq 500/550 High Output Kit v2 150 Cycles (15057931, Illumia) and paired-end sequenced in a Nextseq 550 (Illumina).
Project description:Description: SMARCD1, an auxiliary subunit of the SWI/SNF complex is essential for maintaining embryonic stem cell pluripotency and metabolic gene regulation. Here, we have tried to address the role of SMARCD1 in myeloid differentiation and leukemia. To address this, we transduced HL60 cells with lentiviral particles expressing shcontrol and shSMARCD1. Next we subjected these cells to 10 nM vitamin D3 induction for 48 h. RNA-sequencing was conducted for shcontrol and shSMARCD1 in uninduced and vitamin D3 induced HL-60 cells. High quality RNA with RIN values above 9, was used to generate library using TruSeq™ stranded mRNA kit (20020595, Illumina) following manufacturer’s instructions. Finally barcoded libraries were pooled, and a final concentration of 1.2 pM was loaded using NextSeq 500/550 High Output Kit v2 150 Cycles (15057931, Illumia) and paired-end sequenced in a Nextseq 550 (Illumina).
Project description:To identify candidates of interest that were more highly expressed in BAT than WAT, we conducted RNAseq in human primary brown and white adipocytes. Adipose tissue was obtained from the central compartment of the neck, superior to the clavicle and deep to the lateral thyroid lobe either adjacent to the longus colli muscle or to the oesophagus (brown adipose tissue) and more superficially from the subcutaneous neck tissue (white adipose tissue). The stromal vascular fraction was isolated and cultured as described (Ramage, Akyol et al. 2016 doi: 10.1016/j.cmet.2016.06.011). Following differentiation, cells were cultured in serum-stripped medium for 48 hours prior to RNA extraction and subsequent bulk RNA-seq.
Project description:RNAseq of enterocytes of the jejunum and Ileum from transgene C57BL/6 mice to assess the effects of a CKIα knock-out in combination with expression of a p53 mutant protein (R172H ) compared to a double knock-out of CKIα and p53. In addition, a group of mice exhibiting a CKIα knock-out and mutant p53 (R172H) was treated with gallic acid and compared to untreated mice (Jejunum only).
Project description:Cornelia de Lange syndrome (CdLS) is a rare genetic disease associated with cohesinopathy. A novel iPSC line was generated from the CdLS patient carrying a heterozygous missense point-mutation of the NIPBL gene. iPSC lines prepared from the healthy parents and the mutation-corrected isogenic cell lines are used as the respective controls. Upon differentiation into hepatocytes, the patient-derived iPSC demonstrate the capacity to express hepatocyte-specific marker transcripts and the respective proteins, however, the efficiency of differentiation is significantly inferior to those of the respective controls, demonstrated by single-cell RNA sequencing and immune-fluorescent analyses. Global change of transcriptome in the patient-derived iPSC relative to the control cells is associated with altered chromatin accessibility, assessed by RNAseq combined with ATACseq analysis. Differentially down-regulated genes in the patient-derived iPSC, in particular, are those coding for proteins related to neural differentiation and transcription factors, while up-regulated genes contain some of antisense RNA coding genes, pseudo genes and long non-coding RNAs. Some of the differentially regulated genes are consistent with the altered chromatin accessibility and this observation is consistent during hepatocyte differentiation. Thus, the mutation in the NIPBL gene of the CdLS patient could be responsible for defects of differentiation primarily due to alteration of chromatin accessibility.
Project description:Upon virus infections, the transcriptomic profile of host plants markedly changes. The rapid and comprehensive transcriptional reprogramming is critical to ward off virus attack. To learn more about transcriptional reprogramming in tobamovirus-infected pepper leaves, we carried out transcriptome-wide RNA-Seq analyses of pepper leaves following Obuda pepper virus (ObPV) and Pepper mild mottle virus (PMMoV)-inoculations.
Project description:To study expression pattern of small nucleolar RNAs (snoRNAs) during influenza A viral infection, human cells A549 were infected with influenza A/Puerto Rico/8/1934 (H1N1) virus. Small RNA-seq analysis of infected cells after 24 h or 48 h incubation was performed on an Illumina NextSeq 500 platform. The same mock-infected cells were used as control. Small RNA fractions (<200 nucleotide length) was used for constructing of cDNA libraries. Differential expressed non-coding RNAs were identified using R package DESeq2.
Project description:In this study monoclonal cell lines carrying mutations in the small nucleolar RNA host-gene GAS5 were obtained. Next, Poly(A) RNA-seq and Small RNA-seq analyses of obtained cell lines were performed on an Illumina NextSeq 500 platform. Poly(A) RNA-seq libraries was constructed on polyA mRNA-enriched fraction. Small RNA-seq libraries was constructed on small RNA fraction (<200 nucleotide length). The reads were aligned via STAR to the human genome (GRCh37). The comparison of the mRNA levels of genes and small RNA levels in RNA-Seq experiments was carried out using CuffDiff tool. Sashimi plots for RNA-Seq analyses of isoform expression were generated by IGV. It was shown that specific mutations in SNORD74 led to the downregulation of all GAS5-encoded SNORDs and GAS5 lncRNA. Obtained results demonstrate the SNORD-dependent manner of the GAS5 maturation.
Project description:Ppm1d T/+ mice exhibit altered BM composition. The aim is to determine the molecular mechanisms possibly responsible for this alterations in sorted long-term hematopoietic stem cells isolated from bone marrow. Long-term HSCs (LT-HSCs sorted from 12-week-old WT and Ppm1DT/+ mice. The Ppm1dT/+ HSC transcriptome shows enrichment of MYC, mTOR compared to WT.