Chromatin accessibility and microRNA expression in nephron progenitor cells during kidney development [ATAC-Seq]
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ABSTRACT: Mammalian nephron progenitors undergo transcriptional changes over the course of nephrogenesis that sensitize them to signals for differentiation over time. This increases the rate at which they exit their multipotent, self-renewing mesenchymal state and become differentiated epithelial cells, ultimately depleting the progenitor population and leading to the cessation of nephrogenesis. We hypothesized that changes in chromain accessibility and miRNA expression would accompany these transcriptional changes, and that regions of changing chromatin accessibiltiy could reveal chaning regulatory features such as enhancers, including some that affect miRNA expression. To test this, we pooled kidneys from wild-type mouse litters sacrificed at embryonic day 14.5 (E14.5) and post-natal day zero (P0) using positive selection for the surface protein Integrin alpha 8 (Itga8), then sequenced the assay for transposase-accessible chromatin (ATAC-seq) using 50,000 cells, and used the remaining cells for small RNA sequencing (smRNA-seq). ATAC-seq libraries were generated using the Nextera DNA Flex Library Prep Kit (Illumina FC-121-1030) with modifications according to a previously published method (Buenrostro et al., 2013), and all sequencing was performed using an Illumina NextSeq500 by the Health Sciences Sequencing Core at UPMC Children's Hospital of Pittsburgh.
Project description:Mammalian nephron progenitors undergo transcriptional changes over the course of nephrogenesis that sensitize them to signals for differentiation over time. This increases the rate at which they exit their multipotent, self-renewing mesenchymal state and become differentiated epithelial cells, ultimately depleting the progenitor population and leading to the cessation of nephrogenesis. We hypothesized that changes in chromain accessibility and miRNA expression would accompany these transcriptional changes, and that regions of changing chromatin accessibiltiy could reveal chaning regulatory features such as enhancers, including some that affect miRNA expression. To test this, we pooled kidneys from wild-type mouse litters sacrificed at embryonic day 14.5 (E14.5) and post-natal day zero (P0) using positive selection for the surface protein Integrin alpha 8 (Itga8), then sequenced the assay for transposase-accessible chromatin (ATAC-seq) using 50,000 cells, and used the remaining cells for small RNA sequencing (smRNA-seq). smRNA-seq libraries were generated using the QIAseq miRNA library preparation kit (Qiagen 331502), and all sequencing was performed using an Illumina NextSeq500 by the Health Sciences Sequencing Core at UPMC Children's Hospital of Pittsburgh.
Project description:Open chromatin regions were analyzed by ATAC-seq in t(3;8) K562 and wild type K562 to characterize the MYC super-enhancer region. ATAC-seq was performed as described (Buenrostro et al., 2013) with a modification in the lysis buffer to reduce mitochondrial DNA contamination.
Project description:Open chromatin regions were analyzed by ATAC-seq in MUTZ3 and MOLM1 cell lines (both inv(3) AML) to characterize the GATA2 super-enhancer region. ATAC-seq was performed as described (Buenrostro et al., 2013) with a modification in the lysis buffer to reduce mitochondrial DNA contamination.
Project description:ATAC-seq of 79 primary samples obtained from human acute leukemias, namely AML, T-ALL and mixed myeloid/lymphoid leukemias with CpG Island Methylator Phenotype (CIMP). Moreover, ATAC-seq of CD34+ HSPCs from 3 healthy donors are included. ATAC-seq was performed as described (Buenrostro et al., 2013) with a modification in the lysis buffer to reduce mitochondrial DNA contamination. Due to patient confidentiality considerations, the raw data files for this dataset have been deposited to the EGA controlled-access archive under the accession numbers EGAS00001007094 (study); EGAD00001011050 (dataset).
Project description:We used ATAC-seq (Buenrostro et al, 2013) to identify regions of open chromatin in FACS sorted mouse endothelial cells from E12.5 hearts. Please note that the animals were injected at different dates, which resulted in different effects of the knockout, as indicated in the batch attribute of the samples. This data set is part of the study \Endocardial Tbx20 is essential for mesenchymal and myocardial cell movements required for cardiac septation\. Peaks were called using HOMER (-gsize 1.87e9 -region -tbp 1). Replicate 1: -fdr 1e-8, replicate 2: default parameters. Buenrostro JD, Giresi PG, Zaba LC, Chang HY, Greenleaf WJ. 2013. Transposition of native chromatin for fast and sensitive epigenomic profiling of open chromatin, DNA-binding proteins and nucleosome position. Nat Methods 10: 12138.
Project description:Gata5/6 are essential regulators in zebrafish heart development. To understand how Gata5/6 affects the chromatin accessibility and regulates cardiac gene expression, we FACS-isolated GFP+ and GFP- cells from Tg(gata5:GFP) transgenic embryos in both control and Gata5/6 deficient embryos (injected with Gata5/6 morpholinos) and conducted ATAC-seq. We performed the experiments at 8 hours post fertilization, intending to dissect the role of Gata5/6 in the earliest step in cardiac lineage specification. We did 2-3 biological replicates for each sample. ATAC-seq libraries were made using previously published protocol (Buenrostro et al., Nat. Methods, 2013. DOI: 10.1038/nmeth.2688) and sequenced on Illumina Hiseq2500.
Project description:Primary human foreskin fibroblasts (HFF) were exposed to either salt stress (80mM KCl) or heat stress (44ºC). ATAC-seq libraries were prepared starting with 50,000 cells per condition following the protocol described by Buenrostro et al., Nature Methods 2013
Project description:Primary human foreskin fibroblasts (HFF) were infected with wild-type simplex virus 1 (HSV-1) strain 17 at a multiplicity of infection (MOI) of 10. ATAC-seq libraries were prepared starting with 50,000 cells per condition following the protocol described by Buenrostro et al., Nature Methods 2013
Project description:Using ChIP-seq of RUNX1 in the cells with cBAF-targeting gRNA, we identified RUNX1 binding chromain loci which is regulated by cBAF complex
Project description:Primary human fetal foreskin fibroblasts (HFFF) were infected with wild-type simplex virus 1 (HSV-1) strain F and null-ICP22 mutant at a multiplicity of infection (MOI) of 10. ATAC-seq libraries were prepared starting with 50,000 cells per condition following the protocol described by Buenrostro et al., Nature Methods 2013