Project description:Transcriptome analysis of total RNA samples from HEK293-PIGS-KO and HEK293-PIGS-UBE2J1-DKO cells. To check whether KO of UBE2J1 upregulates genes of GPI biosthesis pathway, we used microarrays to analyze gene expression change by KO of UBE2J1 and comfirmed that known GPI pathway genes are not changed by ERAD-deficiency.
Project description:RNA Sequencing of H1 WT hESCs, H1 QSER1 KO hESCs, H1 TET1 KO hESCs, H1 QSER1/TET1 DKO hESCs, WT Day10 embryoid bodies (EBs), QSER1 KO Day10 EBs, TET1 KO Day10 EBs, QSER1/TET1 DKO Day10 EBs, WT pancreatic progenitors (PP1), QSER1 KO PP1, TET1 KO PP1, and QSER1/TET1 DKO PP1. DNA methylation is essential to mammalian development, and dysregulation can cause serious pathological conditions. Key enzymes responsible for deposition and removal of DNA methylation are known, but how they cooperate to tightly regulate the methylation landscape remains a central question. Utilizing a knockin DNA methylation reporter, we performed a genome-wide CRISPR/Cas screen in human embryonic stem cells to discover DNA methylation regulators. The top screen hit was an uncharacterized gene QSER1, which proved to be a key guardian of bivalent promoters and poised enhancers of developmental genes, especially those residing in DNA methylation valleys (or canyons). We further demonstrate cooperation of QSER1 and TET1 through genetic and biochemical interactions to inhibit DNMT3-mediated de novo methylation and safeguard developmental programs.
Project description:To characterize the relationship between IRF5 and Lyn in innate immune responses at a genome-wide scale, we performed gene expression microarray analysis for wild-type (WT), Irf5-KO, Lyn-KO and Lyn/Irf5-DKO bone marrow-derived dendritic cells (BMDCs) with or without 6 h-stimulation with 150 nM CpG-B ODN. We extracted the transcripts that were upregulated by CpG-B ODN in WT BMDCs, further upregulated in Lyn-KO BMDCs and then downregulated in Lyn/Irf5-DKO BMDCs (fold change â?¥ 2, false discovery rate < 0.05). When these 79 transcripts were sorted by their expression levels in Lyn-KO BMDCs, most of the top 20 genes (13 out of 20) were those encoding type I interferons (IFNs), indicating that Lyn particularly suppresses IRF5 induction of type I IFNs. BMDCs from WT, Irf5-KO, Lyn-KO and Lyn/Irf5-DKO mice in a C57BL/6 background were unstimulated or stimulated with CpG-B ODN. Biological triplicate for each genotype were analyzed (24 samples in total).
Project description:Purpose: Next-generation sequencing (NGS) has revolutionized systems-based analysis of cellular pathways. The goals of this study are to compare the genes expression difference at transcriptome level; Methods: Total RNA was extracted from whole cells with the mirVana miRNA Isolation Kit according to the manufacturer’s protocol. RNA quality and integrity were evaluated with an Agilent 2100 Bioanalyzer. Samples with an RNA integrity number (RIN) ≥ 7 were considered to be of high quality and were processed further and subjected to subsequent analysis. Total RNA-seq libraries were generated using 4 μg of total RNA, which was analyzed using the TruSeq Stranded mRNA LTSample Prep Kit. These libraries were then sequenced using the Illumina sequencing platform (HiSeqTM 2500 or Illumina HiSeq X Ten), and 125-bp/150-bp paired-end reads were generated. Results: The raw reads containing adaptors and the low-quality reads from the raw data were removed using Trimmomatic to obtain clean reads. Transcriptome sequencing was conducted by OE Biotech Co., Ltd. (Shanghai, China), and clean reads were provided. The clean reads were mapped to the hg38 reference genome using hisat2 (version 2.1.0). The output BAM files were converted to SAM files using SAMtools 1.9. The final TPM values were obtained using Stringtie 1.3.5. Conclusions: To understand the mechanistic basis of GPI biosynthesis upregulation by the CD55 precursor, we performed RNA- sequencing (RNA-seq) of samples of parental PIGS-HRD1-DKO, PIGS-HRD1-CD55-TKO, and PIGS-HRD1-CD55-TKO stably overexpressed HA-CD55 stably overexpressing cells. Total RNA was extracted and analyzed. The expression profile of GPI biosynthesis -related genes was not significantly affected by CD55.
Project description:We performed bulk RNA-seq and compared complehensive gene expression profiles of kidney organoids induced from wild type, HNF4A-KO, HNF4G-KO, and HNF4A/4G-DKO iPS cell lines.
Project description:To characterize the relationship between IRF5 and Lyn in innate immune responses at a genome-wide scale, we performed gene expression microarray analysis for wild-type (WT), Irf5-KO, Lyn-KO and Lyn/Irf5-DKO bone marrow-derived dendritic cells (BMDCs) with or without 6 h-stimulation with 150 nM CpG-B ODN. We extracted the transcripts that were upregulated by CpG-B ODN in WT BMDCs, further upregulated in Lyn-KO BMDCs and then downregulated in Lyn/Irf5-DKO BMDCs (fold change ≥ 2, false discovery rate < 0.05). When these 79 transcripts were sorted by their expression levels in Lyn-KO BMDCs, most of the top 20 genes (13 out of 20) were those encoding type I interferons (IFNs), indicating that Lyn particularly suppresses IRF5 induction of type I IFNs.