{"database": "metadata", "table": "run_metadata", "rows": [[65567, "SRR15373448", "SRX11676000", "SRS9707982", "SRP331635", "PRJNA753019", "five prime half of specific tRNAs feeds back to promote corresponding tRNA gene transcription in vertebrate embryos [tRNA seq]", "GSE181683", "Transcriptome Analysis", "five primetRFls are small tRNA fragments derived from five prime half of mature tRNAs. However  it is unknown whether five primetRFls could feed back to regulate tRNA biogenesis. Here we show that five primetRFlGly/GCC and five primetRFlGlu/CTC function to promote transcription of corresponding tRNA genes and are essential for vertebrate early embryogenesis. During zebrafish embryogenesis  dynamics of five primetRFlGly/GCC and five primetRFlGlu/CTC levels correlates with that of tRNAGly/GCC and tRNAGlu/CTC levels. Morpholino mediated knockdown of five primetRFlGly/GCC or five primetRFlGlu/CTC down regulates tRNAGly/GCC or tRNAGlu/CTC levels respectively  and causes embryonic lethality that is efficiently rescued by co injection of properly refolded corresponding tRNA. In zebrafish embryos  tRNA:DNA and five primetRFl:DNA hybrids commonly exist on the template strand of tRNA genes. Mechanistically  unstable five primetRFl:DNA hybrid may prevent the formation of transcriptionally inhibitory stable tRNA:DNA hybrids on the same tRNA loci so as to facilitate tRNA genes transcription. The uncovered mechanism may be implicated in other physiological and pathological processes. Overall design: tRNAs from wildtype zebrafish eggs and early embryos were detected by high throughput sequencing. Total RNAs from mature egg  1cell  256c sphere  shield and 24hpf stage embryos were extracted by Trizol reagent. Then  60 120 nt length RNA was enriched by separation and elution from denature gel. RNA was treated with a combination of ALKB protein to remove some modifications before library preparation.", "parent bioproject:PRJNA753013", "pubmed:34797706", null, "tRNA 1cell", "GSM5509227", null, "tissue:Whole Embryo|genotype:WT|strain:TU|developmental stage:1cell", "tRNA 1cell", "Adaptor trimming and quality control were done by FastP Version 0.19.5 and Cutadapt Version 3.4  then 60 100 nt length reads were preserved for down stream analysis. Reads were first mapped to genome GRCz11 by Bowtie2 Version 2.4.2 with very sensitive local mode  D 20  R 3  N 0  L 20  i S 1 0.50. Then aligned reads were further mapped to tRNA database GtRNAdb  GRCz11. The counts of reads aligned to each tRNAs were calculated and normalized to all reads mapped to genome for down stream analysis. Genome build: GRCz11 Genome build: tRNA database from GtRNAdb  GRCz11 Supplementary files format and content: tab delimited text files include tRNA RPM values for each sample", "Whole Embryo", null, "About 50 100 zebrafish eggs or embryos at a desired stage were collected to extract total RNA  and dechorionated by Pronase digestion. Embryos were then transferred to 1.5 ml eppendorf tube  and lysed in 1 ml Trizol Reagent Thermo Fisher Scientific  15596018. The lysate was centrifuged at 12 000 rpm for 10 min  and the supernatant was collected and mixed with 200 \u03bcl chloroform in a fresh tube. post centrifugation at 12 000 rpm for 10 min at 4\u2103  the supernatant was transferred to a fresh tube with addition of equal volume of isopropanol and incubated at room temperature for 15 min  followed by thorough mix and centrifugation at 12 000 rpm for 10 20 min. The RNA pellet was washed by 75% ethanol once and was stored as pellet in 75% ethanol at  80\u2103 until all samples were collected. Total RNA was placed on ice for 30 min with 10% PEG8000 and 0.5 M NaCl  then centrifuged at 14 000 rpm for 15 min at 4\u2103. Supernatant low molecular weight  LMW RNA was collected and precipitated  by addition of ethanol. The LMW RNA was separated by 8% 8 M Urea PAGE gel  then the bands correspond to 60 120 nt length were cut and crunched into small pieces. RNA was eluted from the gel by 0.3 M NaOAc overnight on a vertical rotator at 4\u2103. Then the supernatant was precipitated at  20\u2103 post addition with 2 \u03bcl of Glycoblue and 2.5 volume of ethanol. post centrifuged at 12 000 rpm for 30 min and washed by 75% ethanol  the RNA precipitant was dissolved in RNase free water.  There are three pretreatment steps before library preparation. 60 120nt RNA enriched in the previous step was first incubated in 100 mM Tris HCl pH 9.0 at 37\u2103 for 30 min to be deacylated  then the RNA was treated with T4 PNK to repair ends at 37\u2103 for 30 min. Modifications on tRNAs were removed by ALKB treatment [1 \u03bcg purified RNA was treated by ALKB protein mixture wt ALKB  160 pmol; ALKB D135S  200 pmol; and ALKB D135S/L118V  200 pmol in reaction buffer 300 mM KCl  2 mM MgCl2  50 \u03bcM NH42FeSO42  300 \u03bcM \u03b1 ketoglutarate  2 mM vitamin C  50 \u03bcg/ml BSA  50 mM MES pH 5.0  40 U RNase Inhibitor  1x Protease Inhibitor for 2 h at room temperature]. Purification was needed post each pre treatment step. 100 ng RNA was then subjected for RNA library preparation. tRNA library was prepared by VAHTS Small RNA Library Prep Kit for Illumina Vazyme  NR801. RNA was heat denatured at 80\u2103 for 2 min in the first step  then every steps were done followed the manual in kit. The libraries were first concentrated by DNA concentrator spin column Zymo  then separated on 6% native PAGE gel. Bands correspond to 180 240 bp 60 120 nt RNA ligated with both adaptors length were cut and DNA libraries were eluted from gels.", null, "genotype:WT|strain:TU|developmental stage:1cell", "GSM5509227", "GSM5509227: tRNA 1cell; Danio rerio; RNA Seq", "GSM5509227", null, "1", "About 50 100 zebrafish eggs or embryos at a desired stage were collected to extract total RNA  and dechorionated by Pronase digestion. Embryos were then transferred to 1.5 ml eppendorf tube  and lysed in 1 ml Trizol Reagent Thermo Fisher Scientific  15596018. The lysate was centrifuged at 12 000 rpm for 10 min  and the supernatant was collected and mixed with 200 \u03bcl chloroform in a fresh tube. post centrifugation at 12 000 rpm for 10 min at 4\u2103  the supernatant was transferred to a fresh tube with addition of equal volume of isopropanol and incubated at room temperature for 15 min  followed by thorough mix and centrifugation at 12 000 rpm for 10 20 min. The RNA pellet was washed by 75% ethanol once and was stored as pellet in 75% ethanol at  80\u2103 until all samples were collected. Total RNA was placed on ice for 30 min with 10% PEG8000 and 0.5 M NaCl  then centrifuged at 14 000 rpm for 15 min at 4\u2103. Supernatant low molecular weight  LMW RNA was collected and precipitated  by addition of ethanol. The LMW RNA was separated by 8% 8 M Urea PAGE gel  then the bands correspond to 60 120 nt length were cut and crunched into small pieces. RNA was eluted from the gel by 0.3 M NaOAc overnight on a vertical rotator at 4\u2103. Then the supernatant was precipitated at  20\u2103 post addition with 2 \u03bcl of Glycoblue and 2.5 volume of ethanol. post centrifuged at 12 000 rpm for 30 min and washed by 75% ethanol  the RNA precipitant was dissolved in RNase free water.  There are three pretreatment steps before library preparation. 60 120nt RNA enriched in the previous step was first incubated in 100 mM Tris HCl pH 9.0 at 37\u2103 for 30 min to be deacylated  then the RNA was treated with T4 PNK to repair ends at 37\u2103 for 30 min. Modifications on tRNAs were removed by ALKB treatment [1 \u03bcg purified RNA was treated by ALKB protein mixture wt ALKB  160 pmol; ALKB D135S  200 pmol; and ALKB D135S/L118V  200 pmol in reaction buffer 300 mM KCl  2 mM MgCl2  50 \u03bcM NH42FeSO42  300 \u03bcM \u03b1 ketoglutarate  2 mM vitamin C  50 \u03bcg/ml BSA  50 mM MES pH 5.0  40 U RNase Inhibitor  1x Protease Inhibitor for 2 h at room temperature]. Purification was needed post each pre treatment step. 100 ng RNA was then subjected for RNA library preparation. tRNA library was prepared by VAHTS Small RNA Library Prep Kit for Illumina Vazyme  NR801. RNA was heat denatured at 80\u2103 for 2 min in the first step  then every steps were done followed the manual in kit. The libraries were first concentrated by DNA concentrator spin column Zymo  then separated on 6% native PAGE gel. Bands correspond to 180 240 bp 60 120 nt RNA ligated with both adaptors length were cut and DNA libraries were eluted from gels.", "GEO Accession:GSM5509227", "RNA-Seq", "TRANSCRIPTOMIC", "cDNA", "PAIRED", "ILLUMINA", "Illumina NovaSeq 6000", null, "SRP331635", null, null, "tRNA_1cell_1.fq.gz tRNA_1cell_2.fq.gz", "fastq fastq", 9361966500.0, 31206555.0, "GSM5509227 r1", "0:150 1:150", "A:1533392527;C:1543333302;G:4733567814;T:1550956628;N:716229", 150, 150, null, null, 1533392527, 1543333302, 4733567814, 1550956628, 716229, "SRX11676000", "SRS9707982", "SRA1274130", "GEO", "Anming Meng Lab, School of Life Science, Tsinghua University", 2, 0.7668, 0.00334, 0.06232, 0.00092, 0.81858, 0.99809, 0.57209, 0.77622, 150, 150, "B", "T", "mate2 technical by mapping diff", "illumina", "novaseq_era", "unknown", "small_rna", "unknown", "bulk", "unknown", "unknown", null, "China", "2021-08-09", "Zygote", "Embryo", "Whole Organism", "All anatomical structures"]], "columns": ["rowid", "run.accession", "experiment.accession", "sample.accession", "study.accession", "bioproject", "study.title", "study.alias", "study.type", "study.abstract", "study.attributes", "study.PMIDs", "sample.description", "sample.title", "sample.alias", "sample.centername", "sample.attributes", "GEOsample.title", "GEOsample.dataprocessing", "GEOsample.source", "GEOsample.treatmentprotocol", "GEOsample.extractprotocol", "GEOsample.growthprotocol", "GEOsample.characteristics", "GEOsample.accession", "experiment.title", "experiment.alias", "experiment.library_name", "experiment.design_description", "experiment.library_construction_protocol", "experiment.attributes", "experiment.library_strategy", "experiment.library_source", "experiment.library_selection", "experiment.library_layout", "experiment.platform", "experiment.instrument_model", "experiment.spot_descriptor", "experiment.study_ref", "run.title", "run.attributes", "run.filename", "run.semantic_name", "run.total_bases", "run.total_spots", "run.alias", "run.read_lengths", "run.base_counts", "run.r1_length", "run.r2_length", "run.r3_length", "run.r4_length", "run.Acount", "run.Ccount", "run.Gcount", "run.Tcount", "run.Ncount", "run.experiment", "run.pool_member", "submission.accession", "submission.srasource", "submission.bioprojectsource", "seqdetective.n_mates", "seqdetective.mapping_rate.mate1", "seqdetective.mapping_rate.mate2", "seqdetective.nofeature_rate.mate1", "seqdetective.nofeature_rate.mate2", "seqdetective.sparsity.mate1", "seqdetective.sparsity.mate2", "seqdetective.pos_strand_rate.mate1", "seqdetective.pos_strand_rate.mate2", "seqdetective.readlen.mate1", "seqdetective.readlen.mate2", "seqdetective.judgement.mate1", "seqdetective.judgement.mate2", "seqdetective.judgement.reason", "platform_family", "instrument_generation", "read_bias", "selection_class", "prep_kit", "sc_or_bulk", "tech_class", "technology", "tech_variant", "submission.bioprojectsource.country", "earliest_date", "devstage_curation", "devstage_curation_coarse", "tissue_curation", "tissue_curation_coarse"], "primary_keys": ["rowid"], "primary_key_values": ["65567"], "units": {}, "query_ms": 15.238248000969179}