run_metadata: 60502
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| 60502 | SRR12338629 | SRX8838545 | SRS7101853 | SRP273930 | PRJNA649208 | A zebrafish model of Granulin deficiency reveals essential roles in myeloid cell differentiation | GSE155258 | Transcriptome Analysis | Granulin GRN is a pleiotropic protein involved in inflammation wound healing neurodegenerative disease and tumorigenesis. These roles in human health have prompted research efforts to utilize Granulin in the treatment of rheumatoid arthritis frontotemporal dementia and to enhance wound healing. How granulin contributes to each of these diverse biological functions however remains largely unknown. Here we have uncovered a new role for granulin during myeloid cell differentiation. Using a zebrafish model of granulin deficiency we reveal that myeloid progenitors are unable to terminally differentiate into neutrophils and macrophages in the absence of granulin a grna and fail to express the myeloid genes cebpa rgs2 lyz mpx mpeg1 mfap4 and apoeb. Pathology studies in combination with RNA sequencing show that in addition to facilitating myeloid cell differentiation granulin actively inhibits the erythroid program. Moreover grna deficient myeloid progenitors are incapable of triggering a myelopoiesis emergency response resulting in decreased recruitment of macrophages to the wound and therefore abnormal healing showing aberrant collagen depositions. Mechanistically we have performed CUT&RUN for the first time in zebrafish and identified that Pu.1 directly binds grna enhancers triggering its expression. Similarly mammalian granulin is also upregulated in myeloid cells and its expression is controlled by the myeloid transcription factors PU.1 and IRF8 demonstrating a conserved regulatory mechanism among the zebrafish and mammalian genes. Altogether our findings uncover a previously unrecognized role for granulin during myeloid cell differentiation opening a new field of study that will help elucidate how granulin impacts inflammation wound healing tumor progression and neurodegenerative disease. Overall design: Adult grna / and grna+/+ control fish three fish per condition were subjected to cardiocentesis and kidney dissection as described here. RNA was isolated with RNeasy Qiagen following the manufacturer instructions. Total RNA was assessed for quality using an Agilent Tapestation 4200 and samples with an RNA Integrity Number RIN greater than 8.0 were used to generate RNA sequencing libraries using the TruSeq Stranded mRNA Sample Prep Illumina San Diego CA. Samples were processed following manufacturer's instructions starting with 50 ng of RNA and modifying RNA shear time to five minutes. Resulting libraries were multiplexed and sequenced with 75 basepair bp single reads SR75 to a depth of approximately 20 million reads per sample on an Illumina HiSeq 4000. Samples were demuxltiplexed using bcl2fastq v2.20 Conversion Software Illumina San Diego CA. | AMut3 S16 | GSM4698247 | source name:grna / mutant fish|tissue:kidney marrow|genotype:grna / | AMut3 S16 | RNASeq data was mapped to Reference Consortium Zebrafish Build 10 UCSC Genome GRCz10/danRer10; Sept 2014 using Olego Wu Anczukow et al. 2013 featureCounts Liao Smyth et al. 2014 from subread package is used to compute the raw read counts for each gene. TPM Transcripts Per MillionsLi & Dewey 2011 Pachter 2011 values were computed from the raw read counts using a custom perl script and log2TPM+1 is used to compute the final log reduced expression values. DESeq2 1.26.0 Love Huber et al. 2014 R package is used to compute differentially expressed genes at 1% false discovery rate. Genome build: Reference Consortium Zebrafish Build 10 UCSC Genome GRCz10/danRer10; Sept 2014 Supplementary files format and content: tab delimited text files include TPM values for each Sample. | grna / mutant fish | Six kidney marrows were used to perform RNA seq. One kidney marrow per sample. Triplicates were used per sample: three grna+/+ and three grna / siblings | Zebrafish grna / and grna+/+ control siblings were anesthetized in tricaine subjected to cardiocentesis and kidney dissection as previously described Traver et al. 2003. The resulting kidney suspension was gently triturated with a P1000 pipette and filtered with a 30µm cell strainer. The cell suspension was lysated in RLT buffer and total RNA extraceted using RNeasy Qiagen following the manufacturer instructions. Total RNA was assessed for quality using an Agilent Tapestation 4200 and samples with an RNA Integrity Number RIN greater than 8.0 were used to generate RNA sequencing libraries using the TruSeq Stranded mRNA Sample Prep Illumina San Diego CA. Samples were processed following manufacturer’s instructions starting with 50 ng of RNA and modifying RNA shear time to five minutes. | Zebrafish Danio rerio were raised as described Westerfield 2000 in a circulating aquarium system Aquaneering at 28°C and maintained in accordance with ISU and UCSD IACUC guidelines. | tissue:kidney marrow|genotype:grna / | GSM4698247 | GSM4698247: AMut3 S16; Danio rerio; RNA Seq | GSM4698247 | 1 | Zebrafish grna / and grna+/+ control siblings were anesthetized in tricaine subjected to cardiocentesis and kidney dissection as previously described Traver et al. 2003. The resulting kidney suspension was gently triturated with a P1000 pipette and filtered with a 30µm cell strainer. The cell suspension was lysated in RLT buffer and total RNA extraceted using RNeasy Qiagen following the manufacturer instructions. Total RNA was assessed for quality using an Agilent Tapestation 4200 and samples with an RNA Integrity Number RIN greater than 8.0 were used to generate RNA sequencing libraries using the TruSeq Stranded mRNA Sample Prep Illumina San Diego CA. Samples were processed following manufacturer's instructions starting with 50 ng of RNA and modifying RNA shear time to five minutes. | GEO Accession:GSM4698247 | RNA-Seq | TRANSCRIPTOMIC | cDNA | SINGLE | ILLUMINA | Illumina HiSeq 4000 | SRP273930 | AMut3_S16_L003_R1_001.fastq.gz | fastq | 1660975744.0 | 21854944.0 | GSM4698247 r1 | 0:76 1:0 | A:403133854;C:408666497;G:390547168;T:458402036;N:226189 | 76 | 0 | 403133854 | 408666497 | 390547168 | 458402036 | 226189 | SRX8838545 | SRS7101853 | SRA1104643 | GEO | Boolean, Pediatrics, UCSD | 1 | 0.88856 | 0.071 | 0.7331 | 0.50274 | 76 | B | usable mapping rate | illumina | hiseq_era | unknown | cdna_unspecified | trueseq | bulk | unknown | unknown | United States | 2020-07-28 | Undetermined | Undetermined | Kidney | Renal System |