RNA-seq of RS4:11 cell line populations that are unedited (SPI1 +/+), mono (SPI1 +/-) and biallelicaly edited (SPI1 -/-)
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PROVIDER: PRJEB36256 | ENA |
REPOSITORIES: ENA
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Project description:The goal of the experiment was to identify which genes are differentially expressed between the unedited and SPI1-edited populations. The RS4:11 cell line was edited both mono and biallelicaly via electroporation with guides and Cas9. Following editing, RNA from unedited (SPI1 +/+), mono (SPI1 +/-) and biallellicaly edited (SPI1 -/-) cells were extracted through the Direct-zol RNA Microprep kit. cDNA libraries for sequencing were then prepared using the TruSeq Stranded mRNA Library Prep Kit and the IDT for Illumina-TruSeq RNA UD Indexes (Illumina). Samples were then sequenced on the Illumina NovaSeq platform.
2021-03-24 | E-MTAB-8677 | biostudies-arrayexpress
Project description:The goal of the experiment was to understand the epigenetic effects of PU.1 haploinsufficiency on pro-B cells. The RS4:11 cell line was edited both mono and biallelicaly via electroporation of Cas9 and guides. Following editing, aliquots of unedited (SPI1 +/+), mono (SPI1 +/-) and biallellicaly edited (SPI1 -/-) cells were lysed before undergoing the transposition reaction. After transposition, the ATAC-seq libraries were purified and then amplified via PCR. Libraries were sequenced using the Illumina Novaseq platform.
2021-03-24 | E-MTAB-8676 | biostudies-arrayexpress
Project description:ATAC-seq of RS4:11 cell line populations that are unedited (SPI1 +/+), mono (SPI1 +/-) and biallelicaly edited (SPI1 -/-)
| PRJEB36255 | ENA
Project description:RNA-seq of RS4:11 cell line populations that are unedited (SPI1 +/+), mono (SPI1 +/-) and biallelicaly edited (SPI1 -/-)
2023-06-16 | E-MTAB-8677 | ExpressionAtlas
Project description:The Spi1/ Pu.1 transcription factor plays a crucial role in myeloid cell development across many species. Several Spi1 target genes have been identified so far, yet the Spi1-dependent gene group remains largely unknown. To identify novel genes downstream of Spi1 we employed a microarray strategy using zebrafish embryos. We established the gene group down-regulated upon spi1 knockdown while simultaneously enriched in FACS-sorted embryonic myeloid cells of a spi1:GFP transgenic line, thus representing putative myeloid-specific Spi1 target genes. This gene group contained all previously identified Spi1-dependent zebrafish genes, confirming the validity of the approach, as well as novel immune-related genes. Colocalization studies with neutrophil and macrophage markers revealed that genes cxcr3.2, mpeg1, ptpn6 and mfap4 were expressed specifically in early embryonic macrophages. The analysis of adult zebrafish hematopoietic tissue showed that genes mfap4 and mpeg1 remained macrophage specific within the myeloid fraction throughout zebrafish life. We also demonstrated that gene cxcr3.2, coding for chemokine receptor 3.2, functions in macrophage migration to the site of bacterial infection. These results establish a myeloid-specific gene group dependent on Spi1 in zebrafish and identify novel early macrophage-specific marker genes, which will facilitate further studies of macrophage development and innate immune function.
2010-04-30 | GSE19206 | GEO
Project description:The Spi1/ Pu.1 transcription factor plays a crucial role in myeloid cell development across many species. Several Spi1 target genes have been identified so far, yet the Spi1-dependent gene group remains largely unknown. To identify novel genes downstream of Spi1 we employed a microarray strategy using zebrafish embryos. We established the gene group down-regulated upon spi1 knockdown while simultaneously enriched in FACS-sorted embryonic myeloid cells of a spi1:GFP transgenic line, thus representing putative myeloid-specific Spi1 target genes. This gene group contained all previously identified Spi1-dependent zebrafish genes, confirming the validity of the approach, as well as novel immune-related genes. Colocalization studies with neutrophil and macrophage markers revealed that genes cxcr3.2, mpeg1, ptpn6 and mfap4 were expressed specifically in early embryonic macrophages. The analysis of adult zebrafish hematopoietic tissue showed that genes mfap4 and mpeg1 remained macrophage specific within the myeloid fraction throughout zebrafish life. We also demonstrated that gene cxcr3.2, coding for chemokine receptor 3.2, functions in macrophage migration to the site of bacterial infection. These results establish a myeloid-specific gene group dependent on Spi1 in zebrafish and identify novel early macrophage-specific marker genes, which will facilitate further studies of macrophage development and innate immune function. Zebrafish strains Zebrafish (Danio rerio) were handled in compliance with the local animal welfare regulations and maintained according to standard protocols (http://ZFIN.org). The spi1-gfp transgenic line used in this study (Pu1-lynEGFP, a gift from Franscesca Peri, EMBL, Heidelberg) contains 4 kb of spi1/pu.1 promoter sequence driving expression of membrane-targeted EGFP. Morpholino knock-down experiments Morpholino oligonucleotides (Gene Tools) were diluted to required concentration in 1x Danieu’s buffer (58 mM NaCl, 0.7 mM KCl, 0.4 mM MgSO4, 0.6 mM Ca(NO3)2, 5.0 mM HEPES; pH 7.6) containing 1% Phenol red (Sigma) and approximately 1 nl was injected into the 1-2 cell stage embryo using a Femtojet injector (Eppendorf). Embryo dissociation and FACS (Fluorescent activated cell sorting) Dissociation of embryos was performed according to Covassin et al. 30. In short, ~300 embryos of spi1:GFP line at 28 hpf were dechorionated by pronase treatment, rinsed in calcium free Ringer solution, followed by digestion with 0.25% trypsin. The obtained cell suspension was centrifuged, rinsed with PBS and resuspended in Leibovitz medium L15 without phenol red, 1% fetal calf serum, 0.8mM CaCl2, penicillin 50 U/ml and streptomycin 0.05 mg/ml. The single cell suspension was subject to FACS at room temperature using a FACSAria (Becton Dickinson) with the BD FACSDiva software version 5.0.3 and a Coherent Sapphire solid state laser 488 nm with 13 mW power. The GFP+ and GFP- cell fractions were collected separately into L15 medium supplemented with 0.8 mM CaCl2, 10% fetal calf serum and penicillin 50 U/ml and streptomycin 0.05 mg/ml. The standard yield from circa 300 embryos was approximately 2 x 105 GFP+ cells. Microarray experiment design and analysis Embryos injected either with 1 mM Pu1 morpholino19 or 1 mM Standard Control morpholino were harvested at 28 hours post fertilization (hpf). As a control, embryos injected with an equal volume of 1% Phenol red in Danieu’s buffer (1xPR/D) were used. Pools of 20-30 embryos were collected. RNA samples from spi1 morphants and standard control morphants were labelled with Cy5 and hybridized against a Cy3-labelled common reference (a mixture of all samples injected with 1xPR/D). This experiment was performed in triplicate. FACS sorted spi1-GFP zebrafish embryos at 28hpf were analyzed as follows: the RNA of GFP-positive fraction was labelled with Cy5 and hybridized against a Cy3-labelled RNA of corresponding GFP-negative fraction of cells from the same pool of embryos. This experiment was performed in duplicate. RNA isolation, synthesis of amino allyl labeled aRNA, coupling of Cy3 and Cy5 dyes, and hybridization conditions were as described31. Microarray analysis was performed using custom-designed 44k Agilent chips (platform accession number in GEO: GPL7735) described elsewhere 31. Microarray data were processed and analyzed as described 31. Significance cut-offs for differentially expressed probe sequences were set at 1.2 fold change at P <10-2.
2010-04-30 | E-GEOD-19206 | biostudies-arrayexpress
Project description:Recent small RNA sequencing data has uncovered extensive modification of the 3’ end of mature microRNAs (miRNAs). This non-templated nucleotide addition can impact miRNA gene regulatory networks through the control of miRNA stability or by interfering with the repression of target mRNAs. The miRNA modifying enzymes responsible for this regulation remain largely uncharacterized. Here we describe the ability for two related terminal uridyl transferases (TUTases), Zcchc6 (TUT7) and Zcchc11 (TUT4), to 3’ mono-uridylate a specific subset of miRNAs involved in cell differentiation and Hox gene control. Zcchc6/11 selectively uridylate these miRNAs in vitro, and we biochemically define a bipartite sequence motif that is necessary and sufficient to confer Zcchc6/11 catalyzed uridylation. Depletion of these TUTases in cultured cells causes the selective loss of 3’ mono-uridylation of many of the same miRNAs. Interestingly, upon TUTase dependent loss of uridylation we observe a concomitant increase in non-templated 3’ mono-adenylation. Our results uncover the molecular basis for sequence specific miRNA mono-uridylation by Zcchc6/11, highlight the precise control of different 3’ miRNA modifications in cells, and have implications for miRNA regulation during development.
2013-12-01 | GSE52575 | GEO
Project description:Recent small RNA sequencing data has uncovered extensive modification of the 3’ end of mature microRNAs (miRNAs). This non-templated nucleotide addition can impact miRNA gene regulatory networks through the control of miRNA stability or by interfering with the repression of target mRNAs. The miRNA modifying enzymes responsible for this regulation remain largely uncharacterized. Here we describe the ability for two related terminal uridyl transferases (TUTases), Zcchc6 (TUT7) and Zcchc11 (TUT4), to 3’ mono-uridylate a specific subset of miRNAs involved in cell differentiation and Hox gene control. Zcchc6/11 selectively uridylate these miRNAs in vitro, and we biochemically define a bipartite sequence motif that is necessary and sufficient to confer Zcchc6/11 catalyzed uridylation. Depletion of these TUTases in cultured cells causes the selective loss of 3’ mono-uridylation of many of the same miRNAs. Interestingly, upon TUTase dependent loss of uridylation we observe a concomitant increase in non-templated 3’ mono-adenylation. Our results uncover the molecular basis for sequence specific miRNA mono-uridylation by Zcchc6/11, highlight the precise control of different 3’ miRNA modifications in cells, and have implications for miRNA regulation during development. small RNA profiles in TUTases knock-down and control HeLa cells were generated by Illumina deep sequencing
2013-12-01 | E-GEOD-52575 | biostudies-arrayexpress
Project description:Gene editing using engineered nucleases frequently produces unintended genetic lesions in hematopoietic stem cells (HSCs). Gene-edited HSC cultures thus contain heterogenous populations, the majority of which either do not carry the desired edit or harbor unwanted mutations. In consequence, transplanting edited HSCs carries the risks of suboptimal efficiency and of unwanted mutations in the graft. Here, we present an approach for expanding gene-edited HSCs at clonal density, allowing for genetic profiling of individual clones before transplantation. We achieved this by developing a defined, polymer-based expansion system and identifying long-term expanding clones within the CD201+CD150+CD48-c-Kit+Sca-1+Lin-(KSL) population of pre-cultured HSCs. This dataset compares the gene expression in three different populations: (1) CD201+CD150+CD48-KSL (2) CD201+CD150+CD48+KSL and (3) CD201-KSL cells.
2023-07-19 | GSE232527 | GEO
Project description:Genome wide transcriptional comparison of B. thetaiotaomicron mono-association vs. B. thetaiotaomicron bi-association with B. longum. Cecal populations from 10d colonizations with b6 gnotobiotic mice fed a standard-chow polysaccharide rich (PR) diet (n=5 samples/group) were profiled. Keywords: Mono-association vs. Bi-association, in vivo
2007-01-01 | GSE5866 | GEO