Project description:Bone marrow-derived MDSCs were generated from bone marrow of naive WT or Rel-/- mice. After red blood cell lysis, BM cells were cultured in complete RPMI medium containing GM-CSF (100 ng/mL) and IL-6 (100 ng/mL) for 7 days. RNAs were collected afterwards for RNA-Seq.
Project description:Fibrosarcoma cell lines were generated from wild-type and Tnfaip8 knockout mice. After stimulation with vehicle, LPA, or PDGF, RNAs were collected from the cells afterwards for RNA-Seq.
Project description:The experiment was designed to determine the gene expression changes cultured brown adipocytes in response to the inflammatory stimulus of LPS treatment. Both wild type and TLR4 knockout cells were applied to enable assessment of the contribution of TLR4 to the response.
Project description:The METTL3 methyltransferase is responsible for the deposition of N6-methyladenosine (m6A) modifications in RNA and has been identified as essential for survival and proliferation of acute myeloid leukemia (AML) cells in a genome-wide CRISPR screen. In our experiments involving a small-molecule METTL3 inhibitor (UZH2) in the AML cell line MOLM-13, we observed suppression of cell proliferation, induction of apoptosis and differentiation. The aim of RNA-seq experiment was to characterize the transcriptomic changes occurring in MOLM-13 cell line after treatment UZH2. Cell were treated with 10 µM of UZH2 for 16 h and compered to untreated controls (5 % DMSO).
Project description:To analyse gene expression pattern in different disease state of COVID-19 patients. Experimental workflow: 1) rRNA was removed by using RNase H method, 2) QAIseq FastSelect RNA Removal Kit was used to remove the Globin RNA, 3) The purified fragmented cDNA was combined with End Repair Mix, then add A-Tailing Mix, mix well by pipetting, incubation, 4) PCR amplification, 5) Library quality control and pooling cyclization, 6) The RNA library was sequenced by MGI2000 PE100 platform with 100bp paired-end reads. Analysis steps: 1) RNA-seq raw sequencing reads were filtered by SOAPnuke (Li et al., 2008) to remove reads with sequencing adapter, with low-quality base ratio (base quality < 5) > 20%, and with unknown base (’N’ base) ratio > 5%. 2) Reads aligned to rRNA by Bowtie2 (v2.2.5) (Langmead and Salzberg, 2012) were removed. 3) The clean reads were mapped to the reference genome using HISAT2 (Kim et al., 2015). Bowtie2 (v2.2.5) was applied to align the clean reads to the transcriptome. 4)Then the gene expression level (FPKM) was determined by RSEM (Li and Dewey, 2011). Genes with FPKM > 0.1 in at least one sample were retained.
Project description:Myeloid-derived suppressor cells (MDSCs) are immature myeloid cells that accumulate in the tumor microenvironment of most cancer patients. There MDSCs suppress both adaptive and innate immune responses, hindering immunotherapies. Moreover, many cancers are accompanied by inflammation, a processes that further intensifies MDSC suppressive activity, causing aggressive tumor progression and metastasis. MDSCs collected from tumor-bearing mice profusely release nano-scale membrane-bound extracellular vesicles, called exosomes, which carry biologically active proteins between cells and contribute directly to the immune suppressive functions of MDSC. Many studies on other cell types have shown that exosomes may also carry microRNAs (miRNAs) and messenger RNAs (mRNAs) which can also be transferred to surrounding and distant cells. However, to the best of our knowledge, the miRNA and mRNA cargo of MDSC-derived exosomes has not yet been interrogated. This study aims to identify and quantify the cargo of MDSC and their immunosuppressive exosomes to gather knowledge that can offer insights on the mechanisms by which MDSCs contribute to immune suppression, focusing on the role of exosomes as intercellular communication mediators in the tumor microenvironment. In order to achieve our objective a well-established mouse model based on a conventional mammary carcinoma (4T1 cells) and heightened inflammation (4T1 transduced to express the cytokine interleukin-1b) was used. We provide evidence that MDSC-derived exosomes carry proteins, mRNAs and miRNAs. Relative quantitation demonstrated quantitative differences between the exosome cargo and the cargo of their parental cells, supporting the hypothesis that selective loading into the exosomes is possible. Additionally, quantitative and functional analyses of the exosome cargo generated under conventional and heightened inflammation conditions are consistent with clinical observations that inflammation is linked to cancer development.
Project description:This work aimed to characterize the molecular adaptations occurring in cork oak (Quercus suber) stems in adaptation to drought, and identify key genetic pathways regulating phellem development. One-year-old cork oak plants were grown for additional 6 months under well-watered (WW) or water-deficit (WD) conditions and main stems were targeted for transcriptomic analysis. WD had a negative impact on secondary growth, decreasing the activity of the vascular cambium and phellogen. Following a tissue-specific approach, we analyzed the transcriptional changes imposed by WD in phellem (outer bark), inner bark, and xylem, and found a global downregulation of genes related to cell division, cell wall biogenesis, lignin and/or suberin biosynthesis. Phellem and phloem showed a concerted upregulation of photosynthesis-related genes, suggesting a determinant role of stem photosynthesis in the adaptation of young plants to long-term drought. The data gathered will be important to further harness the diverse genetic background of this species for the development of optimized management practices.
Project description:Smart-seq3xpress was carefully optimized and >1,000 conditions were evaluated. This data submission is organized in 15 datasets that each contain fastq files, unmapped bam files, read count tables, UMI count tables and a barcode annotation file. The barcode_annotation.txt files contain the exact factors/variables tested. Below a short description of each set of experiments: K562_lowvolume: Evaluation of scaling volumes of Smart-seq3 (indicated volume refers to total volume in PCR), whether overlay was used and if cDNA was bead-cleaned or diluted prior to tagmentation. Cell input was K562 cells. The columns \\"treatment\\", \\"volume\\" and \\"VL\\" indicate the experimental parameters. HEK_lowvolume: Evaluation of scaling volumes of Smart-seq3 (indicated volume refers to total volume in PCR), whether overlay was used and if cDNA was bead-cleaned or diluted prior to tagmentation. Cell input was HEK293FT cells. The columns \\"treatment\\", \\"volume\\" and \\"VL\\" indicate the experimental parameters. overlays: Evaluation of the effect of various overlays when generating HEK293FT libraries in 1 uL total volume. The column \\"condition\\" indicates the applied overlay. tagmentations: Evaluation of input cDNA vs Tn5 amount during tagmentation. Purified cDNA from one 384-well plate was used as input into various conditions of tagmentations. The experiments contain evaluation of cDNA amount with fixed Tn5 amount or varying Tn5 amount while keeping the default volume (2 uL) of the tagmentation reaction or scaling the reaction volume. The column \\"condition\\" contains a string indicating reaction volume, cDNA input and Tn5 ATM enzyme amount. If no volume is indicated, reaction was performed in 2 uL. HomeTn5: Evaluation of tagmentation using in-house produced Tn5 enzyme (Picelli et al., 2015) when tagmenting cDNA generated from HEK293FT cells in 1 uL total volume. The column \\"Tn5concentration\\" indicates the Tn5 reaction concentration at 2 uL reaction volume. cycles_cleanups: Optimization of Smart-seq3xpress (column \\"experiment\\" shows \\"direct_tag\\") in regards to clean-ups after cDNA synthesis (column \\"condition\\": noclean, Exo+FastAP, ExoSAP) and dilution volume (9 or 19 uL); PCR cycle numbers (column \\"pcr_input\\") and ATM Tn5 enzyme amount (column \\"ATM\\"). Cell input was HEK293FT cells. PreAmp_Polymerase: Evaluation of various PCR polymerases during initial cDNA amplification. The polymerases are indicated in column \\"polymerase\\". We also evaluated several TSO concentrations (concentration in RT is given) and fwd/rev PCR primers (concentration given in PCR reaction). Cell input was HEK293FT cells. TDE1: Large optimization of tagmentation conditions using the TDE1 Tn5 enzyme. We varied reactions by changing PCR polymerase (KAPA / SeqAmp), PCR extension time and the number of PCR cycles during cDNA amplification. During tagmentation, we varied the amount of TDE1 enzyme, the amount of DMF in the tagmentation reaction buffer and the presence of Tween-20 in the final post-tagmentation PCR. Cell input was HEK293FT cells. TSOs_RT_v1-7: Large scale evaluation of conditions relating to RT and PCR, with a focus on new template-switching oligo (TSO) designs. In total, >20,000 cells and >500 conditions are contained in these datasets. The barcode annotation file contains precise information on the reaction conditions of Lysis, RT, PCR as well as utilized TSO designs. Data was generated from HEK293FT cells and hPBMC (Lonza).
Project description:The study was design to compare transcriptomic profiles of whole biopsies to enteroid/colonoid lines derived from them. In the accompanying publication, we observed substantial overlap of pathways upregulated in Crohn’s disease in enteroids and ileal biopsies, as well as colonoids and rectal biopsies. Our data support the use of patient enteroids and colonoids as critical translational tools for the study of inflammatory bowel disease.
Project description:Approximately 200 cells of Candida albicans lab strain SC5314 were spread on YPD plates supplemented with 2 fold increase from 50 ng/ml to 12800ng/ml caspofungin. All adaptors appeared were collected. Randomly 36 tolerant adaptors were sequenced.