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
0,DRR314108,DRX303511,DRS233566,DRP008373,PRJDB12134,Comparison of expression profile between banp mutant and wildtype sibling.,DRP008373,Other,To characterize the physiological function of Banp the expression profile of banp mutant and wild type sibling was obtained by ATAC sequencing and RNA sequencing. All data were obtained using embryo heads at 48 hpf.,,,,RNA seq of wild type sibling sample3,SAMD00399013,,sample name:rna rw337 48hpf WT rep 3|biological replicate:3,,,,,,,,,Illumina NovaSeq 6000 paired end sequencing of SAMD00399013,DRX303511,1,1,1,,RNA-Seq,TRANSCRIPTOMIC,PolyA,PAIRED,ILLUMINA,Illumina NovaSeq 6000,1510Application ReadForward11Application ReadReverse76,DRP008373,Illumina NovaSeq 6000 paired end sequencing of SAMD00399013,,,,23076492885.0,76679376.0,DRR314108,0:150.51 1:150.44,A:6146969533;C:5373690527;G:5458576301;T:6095818756;N:1437768,150,150,,,6146969533,5373690527,5458576301,6095818756,1437768,DRX303511,DRS233566,DRA012572,OIST|Developmental Neurobiology Unit,Okinawa Institute of Science and Technology,2,0.94223,0.94614,0.10721,0.10288,0.68745,0.68621,0.4728,0.47157,151,150,B,B,biological fallback assumption,illumina,novaseq_era,unknown,poly_a,unknown,bulk,unknown,unknown,,Japan,2022-04-01,Hatching,Embryo,Undetermined,Embryo Imprecise
1,DRR314107,DRX303510,DRS233565,DRP008373,PRJDB12134,Comparison of expression profile between banp mutant and wildtype sibling.,DRP008373,Other,To characterize the physiological function of Banp the expression profile of banp mutant and wild type sibling was obtained by ATAC sequencing and RNA sequencing. All data were obtained using embryo heads at 48 hpf.,,,,RNA seq of wild type sibling sample2,SAMD00399012,,sample name:rna rw337 48hpf WT rep 2|biological replicate:2,,,,,,,,,Illumina NovaSeq 6000 paired end sequencing of SAMD00399012,DRX303510,1,1,1,,RNA-Seq,TRANSCRIPTOMIC,PolyA,PAIRED,ILLUMINA,Illumina NovaSeq 6000,1510Application ReadForward11Application ReadReverse76,DRP008373,Illumina NovaSeq 6000 paired end sequencing of SAMD00399012,,,,26091623771.0,86694066.0,DRR314107,0:150.51 1:150.45,A:6955374552;C:6050013285;G:6169385096;T:6915281392;N:1569446,150,150,,,6955374552,6050013285,6169385096,6915281392,1569446,DRX303510,DRS233565,DRA012572,OIST|Developmental Neurobiology Unit,Okinawa Institute of Science and Technology,2,0.94028,0.94277,0.11038,0.10462,0.68288,0.68134,0.46992,0.47227,150,150,B,B,biological fallback assumption,illumina,novaseq_era,unknown,poly_a,unknown,bulk,unknown,unknown,,Japan,2022-04-01,Hatching,Embryo,Undetermined,Embryo Imprecise
2,DRR314106,DRX303509,DRS233564,DRP008373,PRJDB12134,Comparison of expression profile between banp mutant and wildtype sibling.,DRP008373,Other,To characterize the physiological function of Banp the expression profile of banp mutant and wild type sibling was obtained by ATAC sequencing and RNA sequencing. All data were obtained using embryo heads at 48 hpf.,,,,RNA seq of wild type sibling sample1,SAMD00399011,,sample name:rna rw337 48hpf WT rep 1|biological replicate:1,,,,,,,,,Illumina NovaSeq 6000 paired end sequencing of SAMD00399011,DRX303509,1,1,1,,RNA-Seq,TRANSCRIPTOMIC,PolyA,PAIRED,ILLUMINA,Illumina NovaSeq 6000,1510Application ReadForward11Application ReadReverse76,DRP008373,Illumina NovaSeq 6000 paired end sequencing of SAMD00399011,,,,23833525756.0,79191795.0,DRR314106,0:150.51 1:150.44,A:6324521565;C:5556755459;G:5694025045;T:6256748423;N:1475264,150,150,,,6324521565,5556755459,5694025045,6256748423,1475264,DRX303509,DRS233564,DRA012572,OIST|Developmental Neurobiology Unit,Okinawa Institute of Science and Technology,2,0.94723,0.94975,0.09521,0.09114,0.6776,0.67819,0.46045,0.46153,151,151,B,B,biological fallback assumption,illumina,novaseq_era,unknown,poly_a,unknown,bulk,unknown,unknown,,Japan,2022-04-01,Hatching,Embryo,Undetermined,Embryo Imprecise
3,DRR314105,DRX303508,DRS233563,DRP008373,PRJDB12134,Comparison of expression profile between banp mutant and wildtype sibling.,DRP008373,Other,To characterize the physiological function of Banp the expression profile of banp mutant and wild type sibling was obtained by ATAC sequencing and RNA sequencing. All data were obtained using embryo heads at 48 hpf.,,,,RNA seq of banp mutant sample3,SAMD00399010,,sample name:rna rw337 48hpf Mutant rep 3|biological replicate:3,,,,,,,,,Illumina NovaSeq 6000 paired end sequencing of SAMD00399010,DRX303508,1,1,1,,RNA-Seq,TRANSCRIPTOMIC,PolyA,PAIRED,ILLUMINA,Illumina NovaSeq 6000,1510Application ReadForward11Application ReadReverse76,DRP008373,Illumina NovaSeq 6000 paired end sequencing of SAMD00399010,,,,27688386114.0,92009317.0,DRR314105,0:150.49 1:150.44,A:7681451080;C:6071566134;G:6242782106;T:7690830655;N:1756139,150,150,,,7681451080,6071566134,6242782106,7690830655,1756139,DRX303508,DRS233563,DRA012572,OIST|Developmental Neurobiology Unit,Okinawa Institute of Science and Technology,2,0.91038,0.91556,0.17971,0.16967,0.67718,0.67716,0.47042,0.46425,151,151,B,B,biological fallback assumption,illumina,novaseq_era,unknown,poly_a,unknown,bulk,unknown,unknown,,Japan,2022-04-01,Hatching,Embryo,Undetermined,Embryo Imprecise
4,DRR314104,DRX303507,DRS233562,DRP008373,PRJDB12134,Comparison of expression profile between banp mutant and wildtype sibling.,DRP008373,Other,To characterize the physiological function of Banp the expression profile of banp mutant and wild type sibling was obtained by ATAC sequencing and RNA sequencing. All data were obtained using embryo heads at 48 hpf.,,,,RNA seq of banp mutant sample2,SAMD00399009,,sample name:rna rw337 48hpf Mutant rep 2|biological replicate:2,,,,,,,,,Illumina NovaSeq 6000 paired end sequencing of SAMD00399009,DRX303507,1,1,1,,RNA-Seq,TRANSCRIPTOMIC,PolyA,PAIRED,ILLUMINA,Illumina NovaSeq 6000,1510Application ReadForward11Application ReadReverse76,DRP008373,Illumina NovaSeq 6000 paired end sequencing of SAMD00399009,,,,22970994572.0,76322352.0,DRR314104,0:150.52 1:150.46,A:6142535654;C:5310453740;G:5430124468;T:6086516676;N:1364034,150,150,,,6142535654,5310453740,5430124468,6086516676,1364034,DRX303507,DRS233562,DRA012572,OIST|Developmental Neurobiology Unit,Okinawa Institute of Science and Technology,2,0.93707,0.94063,0.12175,0.11613,0.67825,0.67649,0.46485,0.46905,147,151,B,B,biological fallback assumption,illumina,novaseq_era,unknown,poly_a,unknown,bulk,unknown,unknown,,Japan,2022-04-01,Hatching,Embryo,Undetermined,Embryo Imprecise
5,DRR314103,DRX303506,DRS233561,DRP008373,PRJDB12134,Comparison of expression profile between banp mutant and wildtype sibling.,DRP008373,Other,To characterize the physiological function of Banp the expression profile of banp mutant and wild type sibling was obtained by ATAC sequencing and RNA sequencing. All data were obtained using embryo heads at 48 hpf.,,,,RNA seq of banp mutant sample1,SAMD00399008,,sample name:rna rw337 48hpf Mutant rep 1|biological replicate:1,,,,,,,,,Illumina NovaSeq 6000 paired end sequencing of SAMD00399008,DRX303506,1,1,1,,RNA-Seq,TRANSCRIPTOMIC,PolyA,PAIRED,ILLUMINA,Illumina NovaSeq 6000,1510Application ReadForward11Application ReadReverse76,DRP008373,Illumina NovaSeq 6000 paired end sequencing of SAMD00399008,,,,23637901630.0,78541449.0,DRR314103,0:150.51 1:150.45,A:6359178134;C:5420730443;G:5530700546;T:6325864263;N:1428244,150,150,,,6359178134,5420730443,5530700546,6325864263,1428244,DRX303506,DRS233561,DRA012572,OIST|Developmental Neurobiology Unit,Okinawa Institute of Science and Technology,2,0.9308,0.93545,0.13172,0.12462,0.68219,0.6814,0.46842,0.46984,150,151,B,B,biological fallback assumption,illumina,novaseq_era,unknown,poly_a,unknown,bulk,unknown,unknown,,Japan,2022-04-01,Hatching,Embryo,Undetermined,Embryo Imprecise
6,DRR315802,DRX305194,DRS231989,DRP008318,PRJDB12206,Transcriptome analysis of strip1 mutant and wildtype zebrafish eyes,DRP008318,Transcriptome Analysis,"Strip1 plays essential roles in the developing zebrafish retinal neural circuit. To identify the underlying molecular mechanisms at the transcriptomic level transcriptome of strip1 mutant ""rw147"" eye cups at 2.5 dpf was compared to that of wild type siblings using bulk RNA sequencing analysis.",,,,wildtype sibling sample4,SAMD00400823,,sample name:rw147 2.5dpf wildtype rep 4|biological replicate:4,,,,,,,,,Illumina NovaSeq 6000 paired end sequencing of SAMD00400823,DRX305194,1,1,1,,RNA-Seq,TRANSCRIPTOMIC,PolyA,PAIRED,ILLUMINA,Illumina NovaSeq 6000,1510Application ReadForward11Application ReadReverse76,DRP008318,Illumina NovaSeq 6000 paired end sequencing of SAMD00400823,,,,10262353995.0,34151547.0,DRR315802,0:150.27 1:150.22,A:2735078560;C:2386126821;G:2433638250;T:2707202814;N:307550,150,150,,,2735078560,2386126821,2433638250,2707202814,307550,DRX305194,DRS231989,DRA012640,OIST|Developmental Neurobiology Unit,Developmental Neurobiology Unit,2,0.95231,0.95295,0.09229,0.08773,0.71819,0.72107,0.46746,0.46617,151,150,B,B,biological fallback assumption,illumina,novaseq_era,unknown,poly_a,unknown,bulk,bulk,bulk,,Unknown,2022-03-16,Hatching,Embryo,Undetermined,Embryo Imprecise
7,DRR315801,DRX305193,DRS231988,DRP008318,PRJDB12206,Transcriptome analysis of strip1 mutant and wildtype zebrafish eyes,DRP008318,Transcriptome Analysis,"Strip1 plays essential roles in the developing zebrafish retinal neural circuit. To identify the underlying molecular mechanisms at the transcriptomic level transcriptome of strip1 mutant ""rw147"" eye cups at 2.5 dpf was compared to that of wild type siblings using bulk RNA sequencing analysis.",,,,wildtype sibling sample3,SAMD00400822,,sample name:rw147 2.5dpf wildtype rep 3|biological replicate:3,,,,,,,,,Illumina NovaSeq 6000 paired end sequencing of SAMD00400822,DRX305193,1,1,1,,RNA-Seq,TRANSCRIPTOMIC,PolyA,PAIRED,ILLUMINA,Illumina NovaSeq 6000,1510Application ReadForward11Application ReadReverse76,DRP008318,Illumina NovaSeq 6000 paired end sequencing of SAMD00400822,,,,11516368634.0,38355888.0,DRR315801,0:150.15 1:150.10,A:3080341643;C:2678048339;G:2713051368;T:3044449330;N:477954,150,150,,,3080341643,2678048339,2713051368,3044449330,477954,DRX305193,DRS231988,DRA012640,OIST|Developmental Neurobiology Unit,Developmental Neurobiology Unit,2,0.95353,0.95634,0.08909,0.08533,0.71374,0.71252,0.45986,0.46059,150,151,B,B,biological fallback assumption,illumina,novaseq_era,unknown,poly_a,unknown,bulk,bulk,bulk,,Unknown,2022-03-16,Hatching,Embryo,Undetermined,Embryo Imprecise
8,DRR315800,DRX305192,DRS231987,DRP008318,PRJDB12206,Transcriptome analysis of strip1 mutant and wildtype zebrafish eyes,DRP008318,Transcriptome Analysis,"Strip1 plays essential roles in the developing zebrafish retinal neural circuit. To identify the underlying molecular mechanisms at the transcriptomic level transcriptome of strip1 mutant ""rw147"" eye cups at 2.5 dpf was compared to that of wild type siblings using bulk RNA sequencing analysis.",,,,wildtype sibling sample2,SAMD00400821,,sample name:rw147 2.5dpf wildtype rep 2|biological replicate:2,,,,,,,,,Illumina NovaSeq 6000 paired end sequencing of SAMD00400821,DRX305192,1,1,1,,RNA-Seq,TRANSCRIPTOMIC,PolyA,PAIRED,ILLUMINA,Illumina NovaSeq 6000,1510Application ReadForward11Application ReadReverse76,DRP008318,Illumina NovaSeq 6000 paired end sequencing of SAMD00400821,,,,8814057148.0,29367513.0,DRR315800,0:150.09 1:150.04,A:2350403211;C:2054073465;G:2083044327;T:2326181188;N:354957,150,150,,,2350403211,2054073465,2083044327,2326181188,354957,DRX305192,DRS231987,DRA012640,OIST|Developmental Neurobiology Unit,Developmental Neurobiology Unit,2,0.95287,0.95643,0.0891,0.08586,0.70309,0.70252,0.46384,0.46281,151,149,B,B,biological fallback assumption,illumina,novaseq_era,unknown,poly_a,unknown,bulk,bulk,bulk,,Unknown,2022-03-16,Hatching,Embryo,Undetermined,Embryo Imprecise
9,DRR315799,DRX305191,DRS231986,DRP008318,PRJDB12206,Transcriptome analysis of strip1 mutant and wildtype zebrafish eyes,DRP008318,Transcriptome Analysis,"Strip1 plays essential roles in the developing zebrafish retinal neural circuit. To identify the underlying molecular mechanisms at the transcriptomic level transcriptome of strip1 mutant ""rw147"" eye cups at 2.5 dpf was compared to that of wild type siblings using bulk RNA sequencing analysis.",,,,wildtype sibling sample1,SAMD00400820,,sample name:rw147 2.5dpf wildtype rep 1|biological replicate:1,,,,,,,,,Illumina NovaSeq 6000 paired end sequencing of SAMD00400820,DRX305191,1,1,1,,RNA-Seq,TRANSCRIPTOMIC,PolyA,PAIRED,ILLUMINA,Illumina NovaSeq 6000,1510Application ReadForward11Application ReadReverse76,DRP008318,Illumina NovaSeq 6000 paired end sequencing of SAMD00400820,,,,10491955578.0,34900682.0,DRR315799,0:150.34 1:150.28,A:2796521111;C:2446218287;G:2483414564;T:2765477785;N:323831,150,150,,,2796521111,2446218287,2483414564,2765477785,323831,DRX305191,DRS231986,DRA012640,OIST|Developmental Neurobiology Unit,Developmental Neurobiology Unit,2,0.9539,0.95646,0.08185,0.07808,0.70025,0.70013,0.44713,0.44987,150,151,B,B,biological fallback assumption,illumina,novaseq_era,unknown,poly_a,unknown,bulk,bulk,bulk,,Unknown,2022-03-16,Hatching,Embryo,Undetermined,Embryo Imprecise
10,DRR315798,DRX305190,DRS231985,DRP008318,PRJDB12206,Transcriptome analysis of strip1 mutant and wildtype zebrafish eyes,DRP008318,Transcriptome Analysis,"Strip1 plays essential roles in the developing zebrafish retinal neural circuit. To identify the underlying molecular mechanisms at the transcriptomic level transcriptome of strip1 mutant ""rw147"" eye cups at 2.5 dpf was compared to that of wild type siblings using bulk RNA sequencing analysis.",,,,strip1 mutant sample4,SAMD00400819,,sample name:rw147 2.5dpf Mutant rep 4|biological replicate:4,,,,,,,,,Illumina NovaSeq 6000 paired end sequencing of SAMD00400819,DRX305190,1,1,1,,RNA-Seq,TRANSCRIPTOMIC,PolyA,PAIRED,ILLUMINA,Illumina NovaSeq 6000,1510Application ReadForward11Application ReadReverse76,DRP008318,Illumina NovaSeq 6000 paired end sequencing of SAMD00400819,,,,9197802250.0,30604326.0,DRR315798,0:150.30 1:150.24,A:2468967963;C:2130949980;G:2158692262;T:2438931017;N:261028,150,150,,,2468967963,2130949980,2158692262,2438931017,261028,DRX305190,DRS231985,DRA012640,OIST|Developmental Neurobiology Unit,Developmental Neurobiology Unit,2,0.95159,0.95439,0.10146,0.09758,0.71995,0.71983,0.46519,0.46797,150,151,B,B,biological fallback assumption,illumina,novaseq_era,unknown,poly_a,unknown,bulk,bulk,bulk,,Unknown,2022-03-16,Hatching,Embryo,Undetermined,Embryo Imprecise
11,DRR315797,DRX305189,DRS231984,DRP008318,PRJDB12206,Transcriptome analysis of strip1 mutant and wildtype zebrafish eyes,DRP008318,Transcriptome Analysis,"Strip1 plays essential roles in the developing zebrafish retinal neural circuit. To identify the underlying molecular mechanisms at the transcriptomic level transcriptome of strip1 mutant ""rw147"" eye cups at 2.5 dpf was compared to that of wild type siblings using bulk RNA sequencing analysis.",,,,strip1 mutant sample3,SAMD00400818,,sample name:rw147 2.5dpf Mutant rep 3|biological replicate:3,,,,,,,,,Illumina NovaSeq 6000 paired end sequencing of SAMD00400818,DRX305189,1,1,1,,RNA-Seq,TRANSCRIPTOMIC,PolyA,PAIRED,ILLUMINA,Illumina NovaSeq 6000,1510Application ReadForward11Application ReadReverse76,DRP008318,Illumina NovaSeq 6000 paired end sequencing of SAMD00400818,,,,10498982078.0,34931731.0,DRR315797,0:150.31 1:150.25,A:2804535103;C:2445295179;G:2478768789;T:2770066062;N:316945,150,150,,,2804535103,2445295179,2478768789,2770066062,316945,DRX305189,DRS231984,DRA012640,OIST|Developmental Neurobiology Unit,Developmental Neurobiology Unit,2,0.95448,0.95652,0.0939,0.0887,0.71796,0.71847,0.46335,0.46615,151,151,B,B,biological fallback assumption,illumina,novaseq_era,unknown,poly_a,unknown,bulk,bulk,bulk,,Unknown,2022-03-16,Hatching,Embryo,Undetermined,Embryo Imprecise
12,DRR315796,DRX305188,DRS231983,DRP008318,PRJDB12206,Transcriptome analysis of strip1 mutant and wildtype zebrafish eyes,DRP008318,Transcriptome Analysis,"Strip1 plays essential roles in the developing zebrafish retinal neural circuit. To identify the underlying molecular mechanisms at the transcriptomic level transcriptome of strip1 mutant ""rw147"" eye cups at 2.5 dpf was compared to that of wild type siblings using bulk RNA sequencing analysis.",,,,strip1 mutant sample2,SAMD00400817,,sample name:rw147 2.5dpf Mutant rep 2|biological replicate:2,,,,,,,,,Illumina NovaSeq 6000 paired end sequencing of SAMD00400817,DRX305188,1,1,1,,RNA-Seq,TRANSCRIPTOMIC,PolyA,PAIRED,ILLUMINA,Illumina NovaSeq 6000,1510Application ReadForward11Application ReadReverse76,DRP008318,Illumina NovaSeq 6000 paired end sequencing of SAMD00400817,,,,9850145990.0,32782079.0,DRR315796,0:150.26 1:150.21,A:2636205537;C:2286508705;G:2319481100;T:2607600624;N:350024,150,150,,,2636205537,2286508705,2319481100,2607600624,350024,DRX305188,DRS231983,DRA012640,OIST|Developmental Neurobiology Unit,Developmental Neurobiology Unit,2,0.95193,0.95472,0.09722,0.09375,0.7138,0.71299,0.45542,0.45994,151,151,B,B,biological fallback assumption,illumina,novaseq_era,unknown,poly_a,unknown,bulk,bulk,bulk,,Unknown,2022-03-16,Hatching,Embryo,Undetermined,Embryo Imprecise
13,DRR315795,DRX305187,DRS231982,DRP008318,PRJDB12206,Transcriptome analysis of strip1 mutant and wildtype zebrafish eyes,DRP008318,Transcriptome Analysis,"Strip1 plays essential roles in the developing zebrafish retinal neural circuit. To identify the underlying molecular mechanisms at the transcriptomic level transcriptome of strip1 mutant ""rw147"" eye cups at 2.5 dpf was compared to that of wild type siblings using bulk RNA sequencing analysis.",,,,strip1 mutant sample1,SAMD00400816,,sample name:rw147 2.5dpf Mutant rep 1|biological replicate:1,,,,,,,,,Illumina NovaSeq 6000 paired end sequencing of SAMD00400816,DRX305187,1,1,1,,RNA-Seq,TRANSCRIPTOMIC,PolyA,PAIRED,ILLUMINA,Illumina NovaSeq 6000,1510Application ReadForward11Application ReadReverse76,DRP008318,Illumina NovaSeq 6000 paired end sequencing of SAMD00400816,,,,9542039835.0,31780260.0,DRR315795,0:150.15 1:150.10,A:2543384204;C:2224374632;G:2258183435;T:2515655339;N:442225,150,150,,,2543384204,2224374632,2258183435,2515655339,442225,DRX305187,DRS231982,DRA012640,OIST|Developmental Neurobiology Unit,Developmental Neurobiology Unit,2,0.9528,0.95591,0.08656,0.0828,0.70352,0.70331,0.45316,0.44914,151,151,B,B,biological fallback assumption,illumina,novaseq_era,unknown,poly_a,unknown,bulk,bulk,bulk,,Unknown,2022-03-16,Hatching,Embryo,Undetermined,Embryo Imprecise
303,DRR224539,DRX214824,DRS236347,DRP008458,PRJDB9741,RNA seq for developing pectoral fin in zebrafish,DRP008458,Other,From the developmental view of fin to limb transition an important event in vertebrate evolution we seek fish specific genes that show characteristic expression pattern in the developing fin.,,,,pectoral fin bud from gM1116A zebrafish at 48hpf C,SAMD00222570,,sample name:48hpf C,,,,,,,,,Illumina NovaSeq 6000 paired end sequencing of SAMD00222570,DRX214824,48hpf C,1,Illumina TruSeq Stranded mRNA Library Prep Kit,,RNA-Seq,TRANSCRIPTOMIC,PolyA,PAIRED,ILLUMINA,Illumina NovaSeq 6000,2000Application ReadForward11Application ReadReverse101,DRP008458,Illumina NovaSeq 6000 paired end sequencing of SAMD00222570,,,,16828182200.0,84140911.0,DRR224539,0:100 1:100,A:3957959900;C:4467368792;G:4483580218;T:3919146988;N:126302,100,100,,,3957959900,4467368792,4483580218,3919146988,126302,DRX214824,DRS236347,DRA010086,TOHOKUGL|Laboratory of organ morphogenesis,"Graduate School of Life Sciences, Tohoku University",2,0.97277,0.97115,0.05409,0.05185,0.76717,0.76836,0.46592,0.45571,100,100,B,B,biological fallback assumption,illumina,novaseq_era,unknown,poly_a,trueseq,bulk,unknown,unknown,,Japan,2022-04-21,Hatching,Embryo,Fin,Surface Structure
304,DRR224538,DRX214823,DRS236346,DRP008458,PRJDB9741,RNA seq for developing pectoral fin in zebrafish,DRP008458,Other,From the developmental view of fin to limb transition an important event in vertebrate evolution we seek fish specific genes that show characteristic expression pattern in the developing fin.,,,,pectoral fin bud from gM1116A zebrafish at 48hpf B,SAMD00222569,,sample name:48hpf B,,,,,,,,,Illumina NovaSeq 6000 paired end sequencing of SAMD00222569,DRX214823,48hpf B,1,Illumina TruSeq Stranded mRNA Library Prep Kit,,RNA-Seq,TRANSCRIPTOMIC,PolyA,PAIRED,ILLUMINA,Illumina NovaSeq 6000,2000Application ReadForward11Application ReadReverse101,DRP008458,Illumina NovaSeq 6000 paired end sequencing of SAMD00222569,,,,22815822200.0,114079111.0,DRR224538,0:100 1:100,A:5651922913;C:5760379580;G:5844694518;T:5558658503;N:166686,100,100,,,5651922913,5760379580,5844694518,5558658503,166686,DRX214823,DRS236346,DRA010086,TOHOKUGL|Laboratory of organ morphogenesis,"Graduate School of Life Sciences, Tohoku University",2,0.96814,0.96379,0.03526,0.03362,0.71575,0.71697,0.47605,0.47359,100,100,B,B,biological fallback assumption,illumina,novaseq_era,unknown,poly_a,trueseq,bulk,unknown,unknown,,Japan,2022-04-21,Hatching,Embryo,Fin,Surface Structure
305,DRR224537,DRX214822,DRS236345,DRP008458,PRJDB9741,RNA seq for developing pectoral fin in zebrafish,DRP008458,Other,From the developmental view of fin to limb transition an important event in vertebrate evolution we seek fish specific genes that show characteristic expression pattern in the developing fin.,,,,pectoral fin bud from gM1116A zebrafish at 48hpf A,SAMD00222568,,sample name:48hpf A,,,,,,,,,Illumina NovaSeq 6000 paired end sequencing of SAMD00222568,DRX214822,48hpf A,1,Illumina TruSeq Stranded mRNA Library Prep Kit,,RNA-Seq,TRANSCRIPTOMIC,PolyA,PAIRED,ILLUMINA,Illumina NovaSeq 6000,2000Application ReadForward11Application ReadReverse101,DRP008458,Illumina NovaSeq 6000 paired end sequencing of SAMD00222568,,,,13993230400.0,69966152.0,DRR224537,0:100 1:100,A:3484590137;C:3511805255;G:3624285719;T:3372446706;N:102583,100,100,,,3484590137,3511805255,3624285719,3372446706,102583,DRX214822,DRS236345,DRA010086,TOHOKUGL|Laboratory of organ morphogenesis,"Graduate School of Life Sciences, Tohoku University",2,0.96467,0.96428,0.04367,0.04212,0.72853,0.73125,0.49715,0.49652,100,100,B,B,biological fallback assumption,illumina,novaseq_era,unknown,poly_a,trueseq,bulk,unknown,unknown,,Japan,2022-04-21,Hatching,Embryo,Fin,Surface Structure
10309,ERR7179904,ERX6749461,ERS8138767,ERP132743,PRJEB48372,Transcriptomic profiling of clobetasol propionate induced immunosuppression in challenged zebrafish embryos,E-MTAB-11092,Transcriptome Analysis,The aim of this experiment was to develop an approach for the assessment of immunotoxicity in zebrafish embryos Danio rerio using transcriptomic profiling. Clobetasol propionate a highly potent synthetic corticosteroid which is known to have immunosuppressive effects on vertebrates including zebrafish was used for continuous exposure from fertilization until extraction. As immunotoxicity can only be comprehensively assessed when the effect is observed during an acute infection the embryos were injected with a mixture of different pathogen associated molecular patterns PAMPs consisting of Pam3CSK4 synthetic lipopeptide polyI:C synthetic viral dsRNA and flagellin isolated from Bacillus subtilis. Functional gene expression analysis from samples obtained by different kinds and combinations of treatments in relation to appropriate controls is used to identify pathways that are upregulated by artificial infection and suppressed by treatment with clobetasol propionate. In a modified version of the zebrafish embryo toxicity test OECD 236 20 fertilized eggs were exposed to a sub lethal concentration 250 nM of clobetasol propionate for xxx hours under semi static conditions. The test comprised of three treatments with clobetasol propionate and two negative control NC groups and was performed in triplicates. At 46 hpf the eggs were manually dechorinated. At 48 hpf the embryos were immobilized by anaesthetization using 200 mg/mL tricaine. Using microinjection 8 nL of the mixed PAMPs 0.33 µg/mL Pam3CSK4 0.33 µg/mL polyI:C and 0.033 µg/mL flagellin were injected directly into the embryos bloodstream. One of each exposure groups was injected with PAMPs while another one was injected with ultrapure water to control for effects introduced by the injection process. One control group remained completely untreated. post injection the embryos were further incubated for 3 hours before 10 embryos were randomly picked for each sample and pooled for RNA and protein extraction with NucleoSpin RNA/Protein kit Macherey Nagel. RNA quality was assessed with a 2100 Bioanalyzer system Agilent before coding RNA was purified PolyA selection with TruSeq RNA Library Prep Kit v2 and sequenced on an Illumina HiSeq 4000 System Illumina in 50 bp single read mode. Adapter sequences were removed with trimmomatic and sequences were aligned to the D.rerio reference genome GRCz11 with STAR. Counting of feature mapped reads was performed through featureCounts. Library gene count tables were then merged to a single count matrix as input for differential gene expression analysis with DESeq2.,ENA FIRST PUBLIC:2022 02 25|ENA LAST UPDATE:2022 02 25,,Protocols: For each sample 10 zebrafish larvae were randomly picked from the glass well and carefully transferred to a 1.5 ml low binding Eppendorf tube. Once individuals were collected for all samples the supernatant was removed and the tubes were snap frozen in liquid nitrogen. For detailed information about embryonic incubation conditions and husbandry of adult broodstock please refer to the “Growth protocol”. For detailed information about exposure duration and conditions please refer to the “Treatment protocol”. Adult animal husbandry: Wild type zebrafish Danio rerio Strain AB broodstocks were maintained under flow through conditions in 150 L tanks at 26 +/ 2°C on a 12:12 h light/dark cycle. They were fed daily with TetraMin® Tetra Werke Melle Germany main feed ad libitum and nauplii of Artemia salina. The day before test start glass spawning trays with artificial substrate green glass beads stringed on stainless steel wire were placed at the bottom of each tank. post mating and spawning in the morning hours eggs were rinsed with clear Cu reduced water and placed into glass dishes for pre picking. Fish embryo incubation: For each sample 20 fertilized eggs in the early blastula stage were placed in glass petri dishes diameter 6 cm filled with 8 mL of the testing solution or control medium. Embryos were incubated at 27 +/ 1°C on a 14:10 h light/dark cycle. At 24 hpf eggs were inspected while half of the test solution was renewed. Clobetasol propionate CAS: 25122 46 7 was purchased from Sigma Aldrich Taufkirchen Germany. For the test solution preparation first a 5 mM stock solution was prepared in Acetone 99.9% MS grade. This stock was diluted to 250 µM in acetone before 500 µL were added to a 500 mL DURAN glass bottle. To the bottle for the control solution only 500 µL of pure Acetone was added. post complete evaporation of the acetone the bottle was filled up with 500 mL copper reduced tap water. Complete dissolution of precipitated clobetasol propionate was achieved by sonication for 30 minutes Sonorex RK 100H Bandelin Germany. The test solutions were freshly prepared one day prior the start of the experiment and were constantly aerated at room temperature. The glass wells for the incubation of the embryos were pre saturated with the respective test solution overnight and renewed at the beginning of the experiment. Per sample about 40 50 fertilized fish eggs were pre picked in glass petri dishes 10 cm diameter filled with 30 ml of the testing solution. Using an optical binokular microscope 20 embryos of the same early blastula stage were transferred from the pre picking plate into the test glass wells filled with 8 ml of the test solution. Embryos were then incubated as described in the “growth protocol”. Standard flagellin from Bacillus subtilis ≈ 10% purity Pam3CSK4 ≥ 98 % purity and PolyI:C HMW high molecular weight were purchased from InvivoGen SAS Toulouse France. Stock solutions 1 mg/mL for polyI:C and Pam3CSK4; 0.1 mg/mL for flagellin were prepared in provided endotoxin free water aliquoted and stored according to the manufacturer's instructions until further use. At approximately 46 hpf the embryos were manually dechorinated and then further incubated. A mixture of the different PAMPs was prepared by mixing 10 µL of each component resulting in final concentrations of 0.33 mg/mL for polyI:C and Pam3CSK4 and 0.033 mg/mL for flagellin respectively. The device for microinjection was set up using FemtoTip II injection needles with a complementary universal capillary holder Eppendorf Hamburg Germany connected to an IM 300 microinjector Narishige London United Kingdom. Volume calibration was performed according to Sive et al. 2010 using a micrometer slide with 1/100 mm scale Bresser Rhede Germany. Briefly the needles were backfilled with 10 µL PAMP mix or ultra pure water. Injections for volume calibration were made into a drop of immersion oil purred on the micrometer slide the output pressure was constantly kept at 10 psi while the time of injection was adjusted until an injected drop had a diameter of approximately 250 µm. Assuming that the drop forms a perfect sphere this corresponds to a volume of about 8 nL. The procedure described below was performed for each of the five conditions in turn treating the three replicates simultaneously. In order to anaesthetize the embryos tricaine Sigma Aldrich Taufkirchen Germany stock solution 4 mg/mL in 21 mM Tris pH 9 was added to the petri dishes to a final concentration of 200 µg/mL. The immobilized embryos were transferred onto a 1 % agarose plate which was divided into segments one for each replicate. Excess liquid was removed so that the embryos were kept in place by surface tension. Desiccation was prevented by adding liquid if needed. To ensure that the injections could be performed as quickly as possible the embryos were arranged in a row in a way that their dorsal sides pointed to the same direction. The puncture was made on the dorsal side of the yolk sac and the needle was advanced through the yolk until it reached the yolk sac circulation valley on the ventral side of the embryo where the injection was made directly into the bloodstream. post all replicates of a certain condition were processed the embryos were rinsed back into the corresponding dish containing renewed test solution. Total RNA was extracted using the NucleoSpin RNA/Protein kit from Macherey Nagel 740933.250 The pooled and snap frozen larvae were suspended in RP1 buffer and were transferred to a screw cap tubes filled with 0.3 g of Lysing Matrix D MP Biomedicals 6913050 ceramic beads. Tissue homogenization was performed at 5 m/s for 45 s with FastPrep 24© MP Biomedicals 6004500 at room temperature. From here on RNA was extracted according to the manufacturer's protocol. RNA concentration >100 ng/µl was measured using a Nanodrop 2000 spectrophotometer Thermo Scientific. The sample's overall RNA quality and corresponding RIN value was fluorometrically determined with a 2100 Bioanalyzer© Instrument system Agilent G2939BA using the Agilent RNA 6000 Nano Kit© Agilent 5067 1511 according to the manufacturer's protocol. Only samples with a RIN > 8 were used for downstream analysis. Samples were stored at 80°C until they were send to the sequencing facility on dry ice. Sequencing libraries were prepared for each sample from 100 ng/µl total RNA at the sequencing facility “NGS Services for Integrative Genomics” at the University of Göttingen in Germany. According to their standard workflow cDNA libraries were prepared from protein coding mRNA that were purified through PolyA selection using the TruSeq RNA Library Prep Kit v2 Illumina. Libraries were validated using a Fragment Analyzer system Agilent Santa Clara USA before sequencing.,R1287,SAMEA10486022,"Fraunhofer Attract Eco'n'OMICs, Fraunhofer Institute for Molecular Biology and Applied Ecology, Schmallenberg, Germany",ENA first public:2022 01 01|ENA last update:2022 01 01|External Id:SAMEA10486022|INSDC center alias:Fraunhofer Attract Eco'n'OMICs Fraunhofer Institute for Molecular Biology and Applied Ecology Schmallenberg Germany|INSDC center name:Fraunhofer Attract Eco'n'OMICs Fraunhofer Institute for Molecular Biology and Applied Ecology Schmallenberg Germany|INSDC first public:2022 01 01T00:13:13Z|INSDC last update:2022 01 01T00:13:13Z|INSDC status:public|Submitter Id:E MTAB 11092:R1287|age:51|broker name:ArrayExpress|common name:zebrafish|developmental stage:hatching long pec|genotype:wild type genotype|organism part:whole organism|replicate:T6|sample name:E MTAB 11092:R1287|sex:mixed|stimulus:PAMP|strain:AB,,,,,,,,,Illumina HiSeq 4000 sequencing; Transcriptomic profiling of clobetasol propionate induced immunosuppression in challenged zebrafish embryos,E MTAB 11092:R1287 s,R1287 s,Transcriptomic profiling of clobetasol propionate induced immunosuppression in challenged zebrafish embryos,For each sample 10 zebrafish larvae were randomly picked from the glass well and carefully transferred to a 1.5 ml low binding Eppendorf tube. Once individuals were collected for all samples the supernatant was removed and the tubes were snap frozen in liquid nitrogen. For detailed information about embryonic incubation conditions and husbandry of adult broodstock please refer to the Growth protocol. For detailed information about exposure duration and conditions please refer to the Treatment protocol. Adult animal husbandry: Wild type zebrafish Danio rerio Strain AB broodstocks were maintained under flow through conditions in 150 L tanks at 26 +/ 2C on a 12:12 h light/dark cycle. They were fed daily with TetraMin Tetra Werke Melle Germany main feed ad libitum and nauplii of Artemia salina. The day before test start glass spawning trays with artificial substrate green glass beads stringed on stainless steel wire were placed at the bottom of each tank. post mating and spawning in the morning hours eggs were rinsed with clear Cu reduced water and placed into glass dishes for pre picking. Fish embryo incubation: For each sample 20 fertilized eggs in the early blastula stage were placed in glass petri dishes diameter 6 cm filled with 8 mL of the testing solution or control medium. Embryos were incubated at 27 +/ 1C on a 14:10 h light/dark cycle. At 24 hpf eggs were inspected while half of the test solution was renewed. Clobetasol propionate CAS: 25122 46 7 was purchased from Sigma Aldrich Taufkirchen Germany. For the test solution preparation first a 5 mM stock solution was prepared in Acetone 99.9% MS grade. This stock was diluted to 250 M in acetone before 500 L were added to a 500 mL DURAN glass bottle. To the bottle for the control solution only 500 L of pure Acetone was added. post complete evaporation of the acetone the bottle was filled up with 500 mL copper reduced tap water. Complete dissolution of precipitated clobetasol propionate was achieved by sonication for 30 minutes Sonorex RK 100H Bandelin Germany. The test solutions were freshly prepared one day prior the start of the experiment and were constantly aerated at room temperature. The glass wells for the incubation of the embryos were pre saturated with the respective test solution overnight and renewed at the beginning of the experiment. Per sample about 40 50 fertilized fish eggs were pre picked in glass petri dishes 10 cm diameter filled with 30 ml of the testing solution. Using an optical binokular microscope 20 embryos of the same early blastula stage were transferred from the pre picking plate into the test glass wells filled with 8 ml of the test solution. Embryos were then incubated as described in the growth protocol. Standard flagellin from Bacillus subtilis 10% purity Pam3CSK4 98 % purity and PolyI:C HMW high molecular weight were purchased from InvivoGen SAS Toulouse France. Stock solutions 1 mg/mL for polyI:C and Pam3CSK4; 0.1 mg/mL for flagellin were prepared in provided endotoxin free water aliquoted and stored according to the manufacturers instructions until further use. At approximately 46 hpf the embryos were manually dechorinated and then further incubated. A mixture of the different PAMPs was prepared by mixing 10 L of each component resulting in final concentrations of 0.33 mg/mL for polyI:C and Pam3CSK4 and 0.033 mg/mL for flagellin respectively. The device for microinjection was set up using FemtoTip II injection needles with a complementary universal capillary holder Eppendorf Hamburg Germany connected to an IM 300 microinjector Narishige London United Kingdom. Volume calibration was performed according to Sive et al. 2010 using a micrometer slide with 1/100 mm scale Bresser Rhede Germany. Briefly the needles were backfilled with 10 L PAMP mix or ultra pure water. Injections for volume calibration were made into a drop of immersion oil purred on the micrometer slide the output pressure was constantly kept at 10 psi while the time of injection was adjusted until an injected drop had a diameter of approximately 250 m. Assuming that the drop forms a perfect sphere this corresponds to a volume of about 8 nL. The procedure described below was performed for each of the five conditions in turn treating the three replicates simultaneously. In order to anaesthetize the embryos tricaine Sigma Aldrich Taufkirchen Germany stock solution 4 mg/mL in 21 mM Tris pH 9 was added to the petri dishes to a final concentration of 200 g/mL. The immobilized embryos were transferred onto a 1 % agarose plate which was divided into segments one for each replicate. Excess liquid was removed so that the embryos were kept in place by surface tension. Desiccation was prevented by adding liquid if needed. To ensure that the injections could be performed as quickly as possible the embryos were arranged in a row in a way that their dorsal sides pointed to the same direction. The puncture was made on the dorsal side of the yolk sac and the needle was advanced through the yolk until it reached the yolk sac circulation valley on the ventral side of the embryo where the injection was made directly into the bloodstream. post all replicates of a certain condition were processed the embryos were rinsed back into the corresponding dish containing renewed test solution. Total RNA was extracted using the NucleoSpin RNA/Protein kit from Macherey Nagel 740933.250 The pooled and snap frozen larvae were suspended in RP1 buffer and were transferred to a screw cap tubes filled with 0.3 g of Lysing Matrix D MP Biomedicals 6913050 ceramic beads. Tissue homogenization was performed at 5 m/s for 45 s with FastPrep 24 MP Biomedicals 6004500 at room temperature. From here on RNA was extracted according to the manufacturers protocol. RNA concentration >100 ng/l was measured using a Nanodrop 2000 spectrophotometer Thermo Scientific. The samples overall RNA quality and corresponding RIN value was fluorometrically determined with a 2100 Bioanalyzer Instrument system Agilent G2939BA using the Agilent RNA 6000 Nano Kit Agilent 5067 1511 according to the manufacturers protocol. Only samples with a RIN > 8 were used for downstream analysis. Samples were stored at 80C until they were send to the sequencing facility on dry ice. Sequencing libraries were prepared for each sample from 100 ng/l total RNA at the sequencing facility NGS Services for Integrative Genomics at the University of Gttingen in Germany. According to their standard workflow cDNA libraries were prepared from protein coding mRNA that were purified through PolyA selection using the TruSeq RNA Library Prep Kit v2 Illumina. Libraries were validated using a Fragment Analyzer system Agilent Santa Clara USA before sequencing.,Experimental Factor: compound:Clobetasol propionate|Experimental Factor: dose:250|Experimental Factor: stimulus:PAMP,RNA-Seq,TRANSCRIPTOMIC,PolyA,SINGLE,ILLUMINA,Illumina HiSeq 4000,,ERP132743,Illumina HiSeq 4000 sequencing; Transcriptomic profiling of clobetasol propionate induced immunosuppression in challenged zebrafish embryos,ENA FIRST PUBLIC:2022 02 25|ENA LAST UPDATE:2022 02 25|loader:fastq load.py,p946sR1287_sr.fastq.gz,fastq,2134815688.0,42280846.0,E MTAB 11092:R1287,0:50.49,A:565052096;C:502336362;G:482125031;T:582647501;N:2654698,50,,,,565052096,502336362,482125031,582647501,2654698,ERX6749461,ERS8138767,ERA6860152,Fraunhofer Attract Eco,Fraunhofer Attract Eco,1,0.9356,,0.11997,,0.68314,,0.47934,,51,,B,,usable mapping rate,illumina,hiseq_era,unknown,poly_a,trueseq,bulk,unknown,unknown,,Germany,2022-01-01,Hatching,Embryo,Whole Organism,All anatomical structures
10310,ERR7179903,ERX6749460,ERS8138766,ERP132743,PRJEB48372,Transcriptomic profiling of clobetasol propionate induced immunosuppression in challenged zebrafish embryos,E-MTAB-11092,Transcriptome Analysis,The aim of this experiment was to develop an approach for the assessment of immunotoxicity in zebrafish embryos Danio rerio using transcriptomic profiling. Clobetasol propionate a highly potent synthetic corticosteroid which is known to have immunosuppressive effects on vertebrates including zebrafish was used for continuous exposure from fertilization until extraction. As immunotoxicity can only be comprehensively assessed when the effect is observed during an acute infection the embryos were injected with a mixture of different pathogen associated molecular patterns PAMPs consisting of Pam3CSK4 synthetic lipopeptide polyI:C synthetic viral dsRNA and flagellin isolated from Bacillus subtilis. Functional gene expression analysis from samples obtained by different kinds and combinations of treatments in relation to appropriate controls is used to identify pathways that are upregulated by artificial infection and suppressed by treatment with clobetasol propionate. In a modified version of the zebrafish embryo toxicity test OECD 236 20 fertilized eggs were exposed to a sub lethal concentration 250 nM of clobetasol propionate for xxx hours under semi static conditions. The test comprised of three treatments with clobetasol propionate and two negative control NC groups and was performed in triplicates. At 46 hpf the eggs were manually dechorinated. At 48 hpf the embryos were immobilized by anaesthetization using 200 mg/mL tricaine. Using microinjection 8 nL of the mixed PAMPs 0.33 µg/mL Pam3CSK4 0.33 µg/mL polyI:C and 0.033 µg/mL flagellin were injected directly into the embryos bloodstream. One of each exposure groups was injected with PAMPs while another one was injected with ultrapure water to control for effects introduced by the injection process. One control group remained completely untreated. post injection the embryos were further incubated for 3 hours before 10 embryos were randomly picked for each sample and pooled for RNA and protein extraction with NucleoSpin RNA/Protein kit Macherey Nagel. RNA quality was assessed with a 2100 Bioanalyzer system Agilent before coding RNA was purified PolyA selection with TruSeq RNA Library Prep Kit v2 and sequenced on an Illumina HiSeq 4000 System Illumina in 50 bp single read mode. Adapter sequences were removed with trimmomatic and sequences were aligned to the D.rerio reference genome GRCz11 with STAR. Counting of feature mapped reads was performed through featureCounts. Library gene count tables were then merged to a single count matrix as input for differential gene expression analysis with DESeq2.,ENA FIRST PUBLIC:2022 02 25|ENA LAST UPDATE:2022 02 25,,Protocols: For each sample 10 zebrafish larvae were randomly picked from the glass well and carefully transferred to a 1.5 ml low binding Eppendorf tube. Once individuals were collected for all samples the supernatant was removed and the tubes were snap frozen in liquid nitrogen. For detailed information about embryonic incubation conditions and husbandry of adult broodstock please refer to the “Growth protocol”. For detailed information about exposure duration and conditions please refer to the “Treatment protocol”. Adult animal husbandry: Wild type zebrafish Danio rerio Strain AB broodstocks were maintained under flow through conditions in 150 L tanks at 26 +/ 2°C on a 12:12 h light/dark cycle. They were fed daily with TetraMin® Tetra Werke Melle Germany main feed ad libitum and nauplii of Artemia salina. The day before test start glass spawning trays with artificial substrate green glass beads stringed on stainless steel wire were placed at the bottom of each tank. post mating and spawning in the morning hours eggs were rinsed with clear Cu reduced water and placed into glass dishes for pre picking. Fish embryo incubation: For each sample 20 fertilized eggs in the early blastula stage were placed in glass petri dishes diameter 6 cm filled with 8 mL of the testing solution or control medium. Embryos were incubated at 27 +/ 1°C on a 14:10 h light/dark cycle. At 24 hpf eggs were inspected while half of the test solution was renewed. Clobetasol propionate CAS: 25122 46 7 was purchased from Sigma Aldrich Taufkirchen Germany. For the test solution preparation first a 5 mM stock solution was prepared in Acetone 99.9% MS grade. This stock was diluted to 250 µM in acetone before 500 µL were added to a 500 mL DURAN glass bottle. To the bottle for the control solution only 500 µL of pure Acetone was added. post complete evaporation of the acetone the bottle was filled up with 500 mL copper reduced tap water. Complete dissolution of precipitated clobetasol propionate was achieved by sonication for 30 minutes Sonorex RK 100H Bandelin Germany. The test solutions were freshly prepared one day prior the start of the experiment and were constantly aerated at room temperature. The glass wells for the incubation of the embryos were pre saturated with the respective test solution overnight and renewed at the beginning of the experiment. Per sample about 40 50 fertilized fish eggs were pre picked in glass petri dishes 10 cm diameter filled with 30 ml of the testing solution. Using an optical binokular microscope 20 embryos of the same early blastula stage were transferred from the pre picking plate into the test glass wells filled with 8 ml of the test solution. Embryos were then incubated as described in the “growth protocol”. Standard flagellin from Bacillus subtilis ≈ 10% purity Pam3CSK4 ≥ 98 % purity and PolyI:C HMW high molecular weight were purchased from InvivoGen SAS Toulouse France. Stock solutions 1 mg/mL for polyI:C and Pam3CSK4; 0.1 mg/mL for flagellin were prepared in provided endotoxin free water aliquoted and stored according to the manufacturer's instructions until further use. At approximately 46 hpf the embryos were manually dechorinated and then further incubated. A mixture of the different PAMPs was prepared by mixing 10 µL of each component resulting in final concentrations of 0.33 mg/mL for polyI:C and Pam3CSK4 and 0.033 mg/mL for flagellin respectively. The device for microinjection was set up using FemtoTip II injection needles with a complementary universal capillary holder Eppendorf Hamburg Germany connected to an IM 300 microinjector Narishige London United Kingdom. Volume calibration was performed according to Sive et al. 2010 using a micrometer slide with 1/100 mm scale Bresser Rhede Germany. Briefly the needles were backfilled with 10 µL PAMP mix or ultra pure water. Injections for volume calibration were made into a drop of immersion oil purred on the micrometer slide the output pressure was constantly kept at 10 psi while the time of injection was adjusted until an injected drop had a diameter of approximately 250 µm. Assuming that the drop forms a perfect sphere this corresponds to a volume of about 8 nL. The procedure described below was performed for each of the five conditions in turn treating the three replicates simultaneously. In order to anaesthetize the embryos tricaine Sigma Aldrich Taufkirchen Germany stock solution 4 mg/mL in 21 mM Tris pH 9 was added to the petri dishes to a final concentration of 200 µg/mL. The immobilized embryos were transferred onto a 1 % agarose plate which was divided into segments one for each replicate. Excess liquid was removed so that the embryos were kept in place by surface tension. Desiccation was prevented by adding liquid if needed. To ensure that the injections could be performed as quickly as possible the embryos were arranged in a row in a way that their dorsal sides pointed to the same direction. The puncture was made on the dorsal side of the yolk sac and the needle was advanced through the yolk until it reached the yolk sac circulation valley on the ventral side of the embryo where the injection was made directly into the bloodstream. post all replicates of a certain condition were processed the embryos were rinsed back into the corresponding dish containing renewed test solution. Total RNA was extracted using the NucleoSpin RNA/Protein kit from Macherey Nagel 740933.250 The pooled and snap frozen larvae were suspended in RP1 buffer and were transferred to a screw cap tubes filled with 0.3 g of Lysing Matrix D MP Biomedicals 6913050 ceramic beads. Tissue homogenization was performed at 5 m/s for 45 s with FastPrep 24© MP Biomedicals 6004500 at room temperature. From here on RNA was extracted according to the manufacturer's protocol. RNA concentration >100 ng/µl was measured using a Nanodrop 2000 spectrophotometer Thermo Scientific. The sample's overall RNA quality and corresponding RIN value was fluorometrically determined with a 2100 Bioanalyzer© Instrument system Agilent G2939BA using the Agilent RNA 6000 Nano Kit© Agilent 5067 1511 according to the manufacturer's protocol. Only samples with a RIN > 8 were used for downstream analysis. Samples were stored at 80°C until they were send to the sequencing facility on dry ice. Sequencing libraries were prepared for each sample from 100 ng/µl total RNA at the sequencing facility “NGS Services for Integrative Genomics” at the University of Göttingen in Germany. According to their standard workflow cDNA libraries were prepared from protein coding mRNA that were purified through PolyA selection using the TruSeq RNA Library Prep Kit v2 Illumina. Libraries were validated using a Fragment Analyzer system Agilent Santa Clara USA before sequencing.,R1284,SAMEA10486020,"Fraunhofer Attract Eco'n'OMICs, Fraunhofer Institute for Molecular Biology and Applied Ecology, Schmallenberg, Germany",ENA first public:2022 01 01|ENA last update:2022 01 01|External Id:SAMEA10486020|INSDC center alias:Fraunhofer Attract Eco'n'OMICs Fraunhofer Institute for Molecular Biology and Applied Ecology Schmallenberg Germany|INSDC center name:Fraunhofer Attract Eco'n'OMICs Fraunhofer Institute for Molecular Biology and Applied Ecology Schmallenberg Germany|INSDC first public:2022 01 01T00:13:13Z|INSDC last update:2022 01 01T00:13:13Z|INSDC status:public|Submitter Id:E MTAB 11092:R1284|age:51|broker name:ArrayExpress|common name:zebrafish|developmental stage:hatching long pec|genotype:wild type genotype|organism part:whole organism|replicate:T6|sample name:E MTAB 11092:R1284|sex:mixed|stimulus:water|strain:AB,,,,,,,,,Illumina HiSeq 4000 sequencing; Transcriptomic profiling of clobetasol propionate induced immunosuppression in challenged zebrafish embryos,E MTAB 11092:R1284 s,R1284 s,Transcriptomic profiling of clobetasol propionate induced immunosuppression in challenged zebrafish embryos,For each sample 10 zebrafish larvae were randomly picked from the glass well and carefully transferred to a 1.5 ml low binding Eppendorf tube. Once individuals were collected for all samples the supernatant was removed and the tubes were snap frozen in liquid nitrogen. For detailed information about embryonic incubation conditions and husbandry of adult broodstock please refer to the Growth protocol. For detailed information about exposure duration and conditions please refer to the Treatment protocol. Adult animal husbandry: Wild type zebrafish Danio rerio Strain AB broodstocks were maintained under flow through conditions in 150 L tanks at 26 +/ 2C on a 12:12 h light/dark cycle. They were fed daily with TetraMin Tetra Werke Melle Germany main feed ad libitum and nauplii of Artemia salina. The day before test start glass spawning trays with artificial substrate green glass beads stringed on stainless steel wire were placed at the bottom of each tank. post mating and spawning in the morning hours eggs were rinsed with clear Cu reduced water and placed into glass dishes for pre picking. Fish embryo incubation: For each sample 20 fertilized eggs in the early blastula stage were placed in glass petri dishes diameter 6 cm filled with 8 mL of the testing solution or control medium. Embryos were incubated at 27 +/ 1C on a 14:10 h light/dark cycle. At 24 hpf eggs were inspected while half of the test solution was renewed. Clobetasol propionate CAS: 25122 46 7 was purchased from Sigma Aldrich Taufkirchen Germany. For the test solution preparation first a 5 mM stock solution was prepared in Acetone 99.9% MS grade. This stock was diluted to 250 M in acetone before 500 L were added to a 500 mL DURAN glass bottle. To the bottle for the control solution only 500 L of pure Acetone was added. post complete evaporation of the acetone the bottle was filled up with 500 mL copper reduced tap water. Complete dissolution of precipitated clobetasol propionate was achieved by sonication for 30 minutes Sonorex RK 100H Bandelin Germany. The test solutions were freshly prepared one day prior the start of the experiment and were constantly aerated at room temperature. The glass wells for the incubation of the embryos were pre saturated with the respective test solution overnight and renewed at the beginning of the experiment. Per sample about 40 50 fertilized fish eggs were pre picked in glass petri dishes 10 cm diameter filled with 30 ml of the testing solution. Using an optical binokular microscope 20 embryos of the same early blastula stage were transferred from the pre picking plate into the test glass wells filled with 8 ml of the test solution. Embryos were then incubated as described in the growth protocol. Standard flagellin from Bacillus subtilis 10% purity Pam3CSK4 98 % purity and PolyI:C HMW high molecular weight were purchased from InvivoGen SAS Toulouse France. Stock solutions 1 mg/mL for polyI:C and Pam3CSK4; 0.1 mg/mL for flagellin were prepared in provided endotoxin free water aliquoted and stored according to the manufacturers instructions until further use. At approximately 46 hpf the embryos were manually dechorinated and then further incubated. A mixture of the different PAMPs was prepared by mixing 10 L of each component resulting in final concentrations of 0.33 mg/mL for polyI:C and Pam3CSK4 and 0.033 mg/mL for flagellin respectively. The device for microinjection was set up using FemtoTip II injection needles with a complementary universal capillary holder Eppendorf Hamburg Germany connected to an IM 300 microinjector Narishige London United Kingdom. Volume calibration was performed according to Sive et al. 2010 using a micrometer slide with 1/100 mm scale Bresser Rhede Germany. Briefly the needles were backfilled with 10 L PAMP mix or ultra pure water. Injections for volume calibration were made into a drop of immersion oil purred on the micrometer slide the output pressure was constantly kept at 10 psi while the time of injection was adjusted until an injected drop had a diameter of approximately 250 m. Assuming that the drop forms a perfect sphere this corresponds to a volume of about 8 nL. The procedure described below was performed for each of the five conditions in turn treating the three replicates simultaneously. In order to anaesthetize the embryos tricaine Sigma Aldrich Taufkirchen Germany stock solution 4 mg/mL in 21 mM Tris pH 9 was added to the petri dishes to a final concentration of 200 g/mL. The immobilized embryos were transferred onto a 1 % agarose plate which was divided into segments one for each replicate. Excess liquid was removed so that the embryos were kept in place by surface tension. Desiccation was prevented by adding liquid if needed. To ensure that the injections could be performed as quickly as possible the embryos were arranged in a row in a way that their dorsal sides pointed to the same direction. The puncture was made on the dorsal side of the yolk sac and the needle was advanced through the yolk until it reached the yolk sac circulation valley on the ventral side of the embryo where the injection was made directly into the bloodstream. post all replicates of a certain condition were processed the embryos were rinsed back into the corresponding dish containing renewed test solution. Total RNA was extracted using the NucleoSpin RNA/Protein kit from Macherey Nagel 740933.250 The pooled and snap frozen larvae were suspended in RP1 buffer and were transferred to a screw cap tubes filled with 0.3 g of Lysing Matrix D MP Biomedicals 6913050 ceramic beads. Tissue homogenization was performed at 5 m/s for 45 s with FastPrep 24 MP Biomedicals 6004500 at room temperature. From here on RNA was extracted according to the manufacturers protocol. RNA concentration >100 ng/l was measured using a Nanodrop 2000 spectrophotometer Thermo Scientific. The samples overall RNA quality and corresponding RIN value was fluorometrically determined with a 2100 Bioanalyzer Instrument system Agilent G2939BA using the Agilent RNA 6000 Nano Kit Agilent 5067 1511 according to the manufacturers protocol. Only samples with a RIN > 8 were used for downstream analysis. Samples were stored at 80C until they were send to the sequencing facility on dry ice. Sequencing libraries were prepared for each sample from 100 ng/l total RNA at the sequencing facility NGS Services for Integrative Genomics at the University of Gttingen in Germany. According to their standard workflow cDNA libraries were prepared from protein coding mRNA that were purified through PolyA selection using the TruSeq RNA Library Prep Kit v2 Illumina. Libraries were validated using a Fragment Analyzer system Agilent Santa Clara USA before sequencing.,Experimental Factor: compound:Clobetasol propionate|Experimental Factor: dose:250|Experimental Factor: stimulus:water,RNA-Seq,TRANSCRIPTOMIC,PolyA,SINGLE,ILLUMINA,Illumina HiSeq 4000,,ERP132743,Illumina HiSeq 4000 sequencing; Transcriptomic profiling of clobetasol propionate induced immunosuppression in challenged zebrafish embryos,ENA FIRST PUBLIC:2022 02 25|ENA LAST UPDATE:2022 02 25|loader:fastq load.py,p946sR1284_sr.fastq.gz,fastq,2050986060.0,40612707.0,E MTAB 11092:R1284,0:50.50,A:538161680;C:490403596;G:466089424;T:554663872;N:1667488,50,,,,538161680,490403596,466089424,554663872,1667488,ERX6749460,ERS8138766,ERA6860152,Fraunhofer Attract Eco,Fraunhofer Attract Eco,1,0.93753,,0.1231,,0.68479,,0.45919,,51,,B,,usable mapping rate,illumina,hiseq_era,unknown,poly_a,trueseq,bulk,unknown,unknown,,Germany,2022-01-01,Hatching,Embryo,Whole Organism,All anatomical structures
10311,ERR7179902,ERX6749459,ERS8138765,ERP132743,PRJEB48372,Transcriptomic profiling of clobetasol propionate induced immunosuppression in challenged zebrafish embryos,E-MTAB-11092,Transcriptome Analysis,The aim of this experiment was to develop an approach for the assessment of immunotoxicity in zebrafish embryos Danio rerio using transcriptomic profiling. Clobetasol propionate a highly potent synthetic corticosteroid which is known to have immunosuppressive effects on vertebrates including zebrafish was used for continuous exposure from fertilization until extraction. As immunotoxicity can only be comprehensively assessed when the effect is observed during an acute infection the embryos were injected with a mixture of different pathogen associated molecular patterns PAMPs consisting of Pam3CSK4 synthetic lipopeptide polyI:C synthetic viral dsRNA and flagellin isolated from Bacillus subtilis. Functional gene expression analysis from samples obtained by different kinds and combinations of treatments in relation to appropriate controls is used to identify pathways that are upregulated by artificial infection and suppressed by treatment with clobetasol propionate. In a modified version of the zebrafish embryo toxicity test OECD 236 20 fertilized eggs were exposed to a sub lethal concentration 250 nM of clobetasol propionate for xxx hours under semi static conditions. The test comprised of three treatments with clobetasol propionate and two negative control NC groups and was performed in triplicates. At 46 hpf the eggs were manually dechorinated. At 48 hpf the embryos were immobilized by anaesthetization using 200 mg/mL tricaine. Using microinjection 8 nL of the mixed PAMPs 0.33 µg/mL Pam3CSK4 0.33 µg/mL polyI:C and 0.033 µg/mL flagellin were injected directly into the embryos bloodstream. One of each exposure groups was injected with PAMPs while another one was injected with ultrapure water to control for effects introduced by the injection process. One control group remained completely untreated. post injection the embryos were further incubated for 3 hours before 10 embryos were randomly picked for each sample and pooled for RNA and protein extraction with NucleoSpin RNA/Protein kit Macherey Nagel. RNA quality was assessed with a 2100 Bioanalyzer system Agilent before coding RNA was purified PolyA selection with TruSeq RNA Library Prep Kit v2 and sequenced on an Illumina HiSeq 4000 System Illumina in 50 bp single read mode. Adapter sequences were removed with trimmomatic and sequences were aligned to the D.rerio reference genome GRCz11 with STAR. Counting of feature mapped reads was performed through featureCounts. Library gene count tables were then merged to a single count matrix as input for differential gene expression analysis with DESeq2.,ENA FIRST PUBLIC:2022 02 25|ENA LAST UPDATE:2022 02 25,,Protocols: For each sample 10 zebrafish larvae were randomly picked from the glass well and carefully transferred to a 1.5 ml low binding Eppendorf tube. Once individuals were collected for all samples the supernatant was removed and the tubes were snap frozen in liquid nitrogen. For detailed information about embryonic incubation conditions and husbandry of adult broodstock please refer to the “Growth protocol”. For detailed information about exposure duration and conditions please refer to the “Treatment protocol”. Adult animal husbandry: Wild type zebrafish Danio rerio Strain AB broodstocks were maintained under flow through conditions in 150 L tanks at 26 +/ 2°C on a 12:12 h light/dark cycle. They were fed daily with TetraMin® Tetra Werke Melle Germany main feed ad libitum and nauplii of Artemia salina. The day before test start glass spawning trays with artificial substrate green glass beads stringed on stainless steel wire were placed at the bottom of each tank. post mating and spawning in the morning hours eggs were rinsed with clear Cu reduced water and placed into glass dishes for pre picking. Fish embryo incubation: For each sample 20 fertilized eggs in the early blastula stage were placed in glass petri dishes diameter 6 cm filled with 8 mL of the testing solution or control medium. Embryos were incubated at 27 +/ 1°C on a 14:10 h light/dark cycle. At 24 hpf eggs were inspected while half of the test solution was renewed. Clobetasol propionate CAS: 25122 46 7 was purchased from Sigma Aldrich Taufkirchen Germany. For the test solution preparation first a 5 mM stock solution was prepared in Acetone 99.9% MS grade. This stock was diluted to 250 µM in acetone before 500 µL were added to a 500 mL DURAN glass bottle. To the bottle for the control solution only 500 µL of pure Acetone was added. post complete evaporation of the acetone the bottle was filled up with 500 mL copper reduced tap water. Complete dissolution of precipitated clobetasol propionate was achieved by sonication for 30 minutes Sonorex RK 100H Bandelin Germany. The test solutions were freshly prepared one day prior the start of the experiment and were constantly aerated at room temperature. The glass wells for the incubation of the embryos were pre saturated with the respective test solution overnight and renewed at the beginning of the experiment. Per sample about 40 50 fertilized fish eggs were pre picked in glass petri dishes 10 cm diameter filled with 30 ml of the testing solution. Using an optical binokular microscope 20 embryos of the same early blastula stage were transferred from the pre picking plate into the test glass wells filled with 8 ml of the test solution. Embryos were then incubated as described in the “growth protocol”. Standard flagellin from Bacillus subtilis ≈ 10% purity Pam3CSK4 ≥ 98 % purity and PolyI:C HMW high molecular weight were purchased from InvivoGen SAS Toulouse France. Stock solutions 1 mg/mL for polyI:C and Pam3CSK4; 0.1 mg/mL for flagellin were prepared in provided endotoxin free water aliquoted and stored according to the manufacturer's instructions until further use. At approximately 46 hpf the embryos were manually dechorinated and then further incubated. A mixture of the different PAMPs was prepared by mixing 10 µL of each component resulting in final concentrations of 0.33 mg/mL for polyI:C and Pam3CSK4 and 0.033 mg/mL for flagellin respectively. The device for microinjection was set up using FemtoTip II injection needles with a complementary universal capillary holder Eppendorf Hamburg Germany connected to an IM 300 microinjector Narishige London United Kingdom. Volume calibration was performed according to Sive et al. 2010 using a micrometer slide with 1/100 mm scale Bresser Rhede Germany. Briefly the needles were backfilled with 10 µL PAMP mix or ultra pure water. Injections for volume calibration were made into a drop of immersion oil purred on the micrometer slide the output pressure was constantly kept at 10 psi while the time of injection was adjusted until an injected drop had a diameter of approximately 250 µm. Assuming that the drop forms a perfect sphere this corresponds to a volume of about 8 nL. The procedure described below was performed for each of the five conditions in turn treating the three replicates simultaneously. In order to anaesthetize the embryos tricaine Sigma Aldrich Taufkirchen Germany stock solution 4 mg/mL in 21 mM Tris pH 9 was added to the petri dishes to a final concentration of 200 µg/mL. The immobilized embryos were transferred onto a 1 % agarose plate which was divided into segments one for each replicate. Excess liquid was removed so that the embryos were kept in place by surface tension. Desiccation was prevented by adding liquid if needed. To ensure that the injections could be performed as quickly as possible the embryos were arranged in a row in a way that their dorsal sides pointed to the same direction. The puncture was made on the dorsal side of the yolk sac and the needle was advanced through the yolk until it reached the yolk sac circulation valley on the ventral side of the embryo where the injection was made directly into the bloodstream. post all replicates of a certain condition were processed the embryos were rinsed back into the corresponding dish containing renewed test solution. Total RNA was extracted using the NucleoSpin RNA/Protein kit from Macherey Nagel 740933.250 The pooled and snap frozen larvae were suspended in RP1 buffer and were transferred to a screw cap tubes filled with 0.3 g of Lysing Matrix D MP Biomedicals 6913050 ceramic beads. Tissue homogenization was performed at 5 m/s for 45 s with FastPrep 24© MP Biomedicals 6004500 at room temperature. From here on RNA was extracted according to the manufacturer's protocol. RNA concentration >100 ng/µl was measured using a Nanodrop 2000 spectrophotometer Thermo Scientific. The sample's overall RNA quality and corresponding RIN value was fluorometrically determined with a 2100 Bioanalyzer© Instrument system Agilent G2939BA using the Agilent RNA 6000 Nano Kit© Agilent 5067 1511 according to the manufacturer's protocol. Only samples with a RIN > 8 were used for downstream analysis. Samples were stored at 80°C until they were send to the sequencing facility on dry ice. Sequencing libraries were prepared for each sample from 100 ng/µl total RNA at the sequencing facility “NGS Services for Integrative Genomics” at the University of Göttingen in Germany. According to their standard workflow cDNA libraries were prepared from protein coding mRNA that were purified through PolyA selection using the TruSeq RNA Library Prep Kit v2 Illumina. Libraries were validated using a Fragment Analyzer system Agilent Santa Clara USA before sequencing.,R1275,SAMEA10486018,"Fraunhofer Attract Eco'n'OMICs, Fraunhofer Institute for Molecular Biology and Applied Ecology, Schmallenberg, Germany",ENA first public:2022 01 01|ENA last update:2022 01 01|External Id:SAMEA10486018|INSDC center alias:Fraunhofer Attract Eco'n'OMICs Fraunhofer Institute for Molecular Biology and Applied Ecology Schmallenberg Germany|INSDC center name:Fraunhofer Attract Eco'n'OMICs Fraunhofer Institute for Molecular Biology and Applied Ecology Schmallenberg Germany|INSDC first public:2022 01 01T00:13:13Z|INSDC last update:2022 01 01T00:13:13Z|INSDC status:public|Submitter Id:E MTAB 11092:R1275|age:51|broker name:ArrayExpress|common name:zebrafish|developmental stage:hatching long pec|genotype:wild type genotype|organism part:whole organism|replicate:T6|sample name:E MTAB 11092:R1275|sex:mixed|stimulus:PAMP|strain:AB,,,,,,,,,Illumina HiSeq 4000 sequencing; Transcriptomic profiling of clobetasol propionate induced immunosuppression in challenged zebrafish embryos,E MTAB 11092:R1275 s,R1275 s,Transcriptomic profiling of clobetasol propionate induced immunosuppression in challenged zebrafish embryos,For each sample 10 zebrafish larvae were randomly picked from the glass well and carefully transferred to a 1.5 ml low binding Eppendorf tube. Once individuals were collected for all samples the supernatant was removed and the tubes were snap frozen in liquid nitrogen. For detailed information about embryonic incubation conditions and husbandry of adult broodstock please refer to the Growth protocol. For detailed information about exposure duration and conditions please refer to the Treatment protocol. Adult animal husbandry: Wild type zebrafish Danio rerio Strain AB broodstocks were maintained under flow through conditions in 150 L tanks at 26 +/ 2C on a 12:12 h light/dark cycle. They were fed daily with TetraMin Tetra Werke Melle Germany main feed ad libitum and nauplii of Artemia salina. The day before test start glass spawning trays with artificial substrate green glass beads stringed on stainless steel wire were placed at the bottom of each tank. post mating and spawning in the morning hours eggs were rinsed with clear Cu reduced water and placed into glass dishes for pre picking. Fish embryo incubation: For each sample 20 fertilized eggs in the early blastula stage were placed in glass petri dishes diameter 6 cm filled with 8 mL of the testing solution or control medium. Embryos were incubated at 27 +/ 1C on a 14:10 h light/dark cycle. At 24 hpf eggs were inspected while half of the test solution was renewed. Clobetasol propionate CAS: 25122 46 7 was purchased from Sigma Aldrich Taufkirchen Germany. For the test solution preparation first a 5 mM stock solution was prepared in Acetone 99.9% MS grade. This stock was diluted to 250 M in acetone before 500 L were added to a 500 mL DURAN glass bottle. To the bottle for the control solution only 500 L of pure Acetone was added. post complete evaporation of the acetone the bottle was filled up with 500 mL copper reduced tap water. Complete dissolution of precipitated clobetasol propionate was achieved by sonication for 30 minutes Sonorex RK 100H Bandelin Germany. The test solutions were freshly prepared one day prior the start of the experiment and were constantly aerated at room temperature. The glass wells for the incubation of the embryos were pre saturated with the respective test solution overnight and renewed at the beginning of the experiment. Per sample about 40 50 fertilized fish eggs were pre picked in glass petri dishes 10 cm diameter filled with 30 ml of the testing solution. Using an optical binokular microscope 20 embryos of the same early blastula stage were transferred from the pre picking plate into the test glass wells filled with 8 ml of the test solution. Embryos were then incubated as described in the growth protocol. Standard flagellin from Bacillus subtilis 10% purity Pam3CSK4 98 % purity and PolyI:C HMW high molecular weight were purchased from InvivoGen SAS Toulouse France. Stock solutions 1 mg/mL for polyI:C and Pam3CSK4; 0.1 mg/mL for flagellin were prepared in provided endotoxin free water aliquoted and stored according to the manufacturers instructions until further use. At approximately 46 hpf the embryos were manually dechorinated and then further incubated. A mixture of the different PAMPs was prepared by mixing 10 L of each component resulting in final concentrations of 0.33 mg/mL for polyI:C and Pam3CSK4 and 0.033 mg/mL for flagellin respectively. The device for microinjection was set up using FemtoTip II injection needles with a complementary universal capillary holder Eppendorf Hamburg Germany connected to an IM 300 microinjector Narishige London United Kingdom. Volume calibration was performed according to Sive et al. 2010 using a micrometer slide with 1/100 mm scale Bresser Rhede Germany. Briefly the needles were backfilled with 10 L PAMP mix or ultra pure water. Injections for volume calibration were made into a drop of immersion oil purred on the micrometer slide the output pressure was constantly kept at 10 psi while the time of injection was adjusted until an injected drop had a diameter of approximately 250 m. Assuming that the drop forms a perfect sphere this corresponds to a volume of about 8 nL. The procedure described below was performed for each of the five conditions in turn treating the three replicates simultaneously. In order to anaesthetize the embryos tricaine Sigma Aldrich Taufkirchen Germany stock solution 4 mg/mL in 21 mM Tris pH 9 was added to the petri dishes to a final concentration of 200 g/mL. The immobilized embryos were transferred onto a 1 % agarose plate which was divided into segments one for each replicate. Excess liquid was removed so that the embryos were kept in place by surface tension. Desiccation was prevented by adding liquid if needed. To ensure that the injections could be performed as quickly as possible the embryos were arranged in a row in a way that their dorsal sides pointed to the same direction. The puncture was made on the dorsal side of the yolk sac and the needle was advanced through the yolk until it reached the yolk sac circulation valley on the ventral side of the embryo where the injection was made directly into the bloodstream. post all replicates of a certain condition were processed the embryos were rinsed back into the corresponding dish containing renewed test solution. Total RNA was extracted using the NucleoSpin RNA/Protein kit from Macherey Nagel 740933.250 The pooled and snap frozen larvae were suspended in RP1 buffer and were transferred to a screw cap tubes filled with 0.3 g of Lysing Matrix D MP Biomedicals 6913050 ceramic beads. Tissue homogenization was performed at 5 m/s for 45 s with FastPrep 24 MP Biomedicals 6004500 at room temperature. From here on RNA was extracted according to the manufacturers protocol. RNA concentration >100 ng/l was measured using a Nanodrop 2000 spectrophotometer Thermo Scientific. The samples overall RNA quality and corresponding RIN value was fluorometrically determined with a 2100 Bioanalyzer Instrument system Agilent G2939BA using the Agilent RNA 6000 Nano Kit Agilent 5067 1511 according to the manufacturers protocol. Only samples with a RIN > 8 were used for downstream analysis. Samples were stored at 80C until they were send to the sequencing facility on dry ice. Sequencing libraries were prepared for each sample from 100 ng/l total RNA at the sequencing facility NGS Services for Integrative Genomics at the University of Gttingen in Germany. According to their standard workflow cDNA libraries were prepared from protein coding mRNA that were purified through PolyA selection using the TruSeq RNA Library Prep Kit v2 Illumina. Libraries were validated using a Fragment Analyzer system Agilent Santa Clara USA before sequencing.,Experimental Factor: compound:n1|Experimental Factor: stimulus:PAMP,RNA-Seq,TRANSCRIPTOMIC,PolyA,SINGLE,ILLUMINA,Illumina HiSeq 4000,,ERP132743,Illumina HiSeq 4000 sequencing; Transcriptomic profiling of clobetasol propionate induced immunosuppression in challenged zebrafish embryos,ENA FIRST PUBLIC:2022 02 25|ENA LAST UPDATE:2022 02 25|loader:fastq load.py,p946sR1275_sr.fastq.gz,fastq,1157613512.0,22947522.0,E MTAB 11092:R1275,0:50.45,A:299754053;C:277899399;G:266613838;T:309994905;N:3351317,50,,,,299754053,277899399,266613838,309994905,3351317,ERX6749459,ERS8138765,ERA6860152,Fraunhofer Attract Eco,Fraunhofer Attract Eco,1,0.9411,,0.10048,,0.69008,,0.47233,,51,,B,,usable mapping rate,illumina,hiseq_era,unknown,poly_a,trueseq,bulk,unknown,unknown,,Germany,2022-01-01,Hatching,Embryo,Whole Organism,All anatomical structures
10312,ERR7179901,ERX6749458,ERS8138764,ERP132743,PRJEB48372,Transcriptomic profiling of clobetasol propionate induced immunosuppression in challenged zebrafish embryos,E-MTAB-11092,Transcriptome Analysis,The aim of this experiment was to develop an approach for the assessment of immunotoxicity in zebrafish embryos Danio rerio using transcriptomic profiling. Clobetasol propionate a highly potent synthetic corticosteroid which is known to have immunosuppressive effects on vertebrates including zebrafish was used for continuous exposure from fertilization until extraction. As immunotoxicity can only be comprehensively assessed when the effect is observed during an acute infection the embryos were injected with a mixture of different pathogen associated molecular patterns PAMPs consisting of Pam3CSK4 synthetic lipopeptide polyI:C synthetic viral dsRNA and flagellin isolated from Bacillus subtilis. Functional gene expression analysis from samples obtained by different kinds and combinations of treatments in relation to appropriate controls is used to identify pathways that are upregulated by artificial infection and suppressed by treatment with clobetasol propionate. In a modified version of the zebrafish embryo toxicity test OECD 236 20 fertilized eggs were exposed to a sub lethal concentration 250 nM of clobetasol propionate for xxx hours under semi static conditions. The test comprised of three treatments with clobetasol propionate and two negative control NC groups and was performed in triplicates. At 46 hpf the eggs were manually dechorinated. At 48 hpf the embryos were immobilized by anaesthetization using 200 mg/mL tricaine. Using microinjection 8 nL of the mixed PAMPs 0.33 µg/mL Pam3CSK4 0.33 µg/mL polyI:C and 0.033 µg/mL flagellin were injected directly into the embryos bloodstream. One of each exposure groups was injected with PAMPs while another one was injected with ultrapure water to control for effects introduced by the injection process. One control group remained completely untreated. post injection the embryos were further incubated for 3 hours before 10 embryos were randomly picked for each sample and pooled for RNA and protein extraction with NucleoSpin RNA/Protein kit Macherey Nagel. RNA quality was assessed with a 2100 Bioanalyzer system Agilent before coding RNA was purified PolyA selection with TruSeq RNA Library Prep Kit v2 and sequenced on an Illumina HiSeq 4000 System Illumina in 50 bp single read mode. Adapter sequences were removed with trimmomatic and sequences were aligned to the D.rerio reference genome GRCz11 with STAR. Counting of feature mapped reads was performed through featureCounts. Library gene count tables were then merged to a single count matrix as input for differential gene expression analysis with DESeq2.,ENA FIRST PUBLIC:2022 02 25|ENA LAST UPDATE:2022 02 25,,Protocols: For each sample 10 zebrafish larvae were randomly picked from the glass well and carefully transferred to a 1.5 ml low binding Eppendorf tube. Once individuals were collected for all samples the supernatant was removed and the tubes were snap frozen in liquid nitrogen. For detailed information about embryonic incubation conditions and husbandry of adult broodstock please refer to the “Growth protocol”. For detailed information about exposure duration and conditions please refer to the “Treatment protocol”. Adult animal husbandry: Wild type zebrafish Danio rerio Strain AB broodstocks were maintained under flow through conditions in 150 L tanks at 26 +/ 2°C on a 12:12 h light/dark cycle. They were fed daily with TetraMin® Tetra Werke Melle Germany main feed ad libitum and nauplii of Artemia salina. The day before test start glass spawning trays with artificial substrate green glass beads stringed on stainless steel wire were placed at the bottom of each tank. post mating and spawning in the morning hours eggs were rinsed with clear Cu reduced water and placed into glass dishes for pre picking. Fish embryo incubation: For each sample 20 fertilized eggs in the early blastula stage were placed in glass petri dishes diameter 6 cm filled with 8 mL of the testing solution or control medium. Embryos were incubated at 27 +/ 1°C on a 14:10 h light/dark cycle. At 24 hpf eggs were inspected while half of the test solution was renewed. Clobetasol propionate CAS: 25122 46 7 was purchased from Sigma Aldrich Taufkirchen Germany. For the test solution preparation first a 5 mM stock solution was prepared in Acetone 99.9% MS grade. This stock was diluted to 250 µM in acetone before 500 µL were added to a 500 mL DURAN glass bottle. To the bottle for the control solution only 500 µL of pure Acetone was added. post complete evaporation of the acetone the bottle was filled up with 500 mL copper reduced tap water. Complete dissolution of precipitated clobetasol propionate was achieved by sonication for 30 minutes Sonorex RK 100H Bandelin Germany. The test solutions were freshly prepared one day prior the start of the experiment and were constantly aerated at room temperature. The glass wells for the incubation of the embryos were pre saturated with the respective test solution overnight and renewed at the beginning of the experiment. Per sample about 40 50 fertilized fish eggs were pre picked in glass petri dishes 10 cm diameter filled with 30 ml of the testing solution. Using an optical binokular microscope 20 embryos of the same early blastula stage were transferred from the pre picking plate into the test glass wells filled with 8 ml of the test solution. Embryos were then incubated as described in the “growth protocol”. Standard flagellin from Bacillus subtilis ≈ 10% purity Pam3CSK4 ≥ 98 % purity and PolyI:C HMW high molecular weight were purchased from InvivoGen SAS Toulouse France. Stock solutions 1 mg/mL for polyI:C and Pam3CSK4; 0.1 mg/mL for flagellin were prepared in provided endotoxin free water aliquoted and stored according to the manufacturer's instructions until further use. At approximately 46 hpf the embryos were manually dechorinated and then further incubated. A mixture of the different PAMPs was prepared by mixing 10 µL of each component resulting in final concentrations of 0.33 mg/mL for polyI:C and Pam3CSK4 and 0.033 mg/mL for flagellin respectively. The device for microinjection was set up using FemtoTip II injection needles with a complementary universal capillary holder Eppendorf Hamburg Germany connected to an IM 300 microinjector Narishige London United Kingdom. Volume calibration was performed according to Sive et al. 2010 using a micrometer slide with 1/100 mm scale Bresser Rhede Germany. Briefly the needles were backfilled with 10 µL PAMP mix or ultra pure water. Injections for volume calibration were made into a drop of immersion oil purred on the micrometer slide the output pressure was constantly kept at 10 psi while the time of injection was adjusted until an injected drop had a diameter of approximately 250 µm. Assuming that the drop forms a perfect sphere this corresponds to a volume of about 8 nL. The procedure described below was performed for each of the five conditions in turn treating the three replicates simultaneously. In order to anaesthetize the embryos tricaine Sigma Aldrich Taufkirchen Germany stock solution 4 mg/mL in 21 mM Tris pH 9 was added to the petri dishes to a final concentration of 200 µg/mL. The immobilized embryos were transferred onto a 1 % agarose plate which was divided into segments one for each replicate. Excess liquid was removed so that the embryos were kept in place by surface tension. Desiccation was prevented by adding liquid if needed. To ensure that the injections could be performed as quickly as possible the embryos were arranged in a row in a way that their dorsal sides pointed to the same direction. The puncture was made on the dorsal side of the yolk sac and the needle was advanced through the yolk until it reached the yolk sac circulation valley on the ventral side of the embryo where the injection was made directly into the bloodstream. post all replicates of a certain condition were processed the embryos were rinsed back into the corresponding dish containing renewed test solution. Total RNA was extracted using the NucleoSpin RNA/Protein kit from Macherey Nagel 740933.250 The pooled and snap frozen larvae were suspended in RP1 buffer and were transferred to a screw cap tubes filled with 0.3 g of Lysing Matrix D MP Biomedicals 6913050 ceramic beads. Tissue homogenization was performed at 5 m/s for 45 s with FastPrep 24© MP Biomedicals 6004500 at room temperature. From here on RNA was extracted according to the manufacturer's protocol. RNA concentration >100 ng/µl was measured using a Nanodrop 2000 spectrophotometer Thermo Scientific. The sample's overall RNA quality and corresponding RIN value was fluorometrically determined with a 2100 Bioanalyzer© Instrument system Agilent G2939BA using the Agilent RNA 6000 Nano Kit© Agilent 5067 1511 according to the manufacturer's protocol. Only samples with a RIN > 8 were used for downstream analysis. Samples were stored at 80°C until they were send to the sequencing facility on dry ice. Sequencing libraries were prepared for each sample from 100 ng/µl total RNA at the sequencing facility “NGS Services for Integrative Genomics” at the University of Göttingen in Germany. According to their standard workflow cDNA libraries were prepared from protein coding mRNA that were purified through PolyA selection using the TruSeq RNA Library Prep Kit v2 Illumina. Libraries were validated using a Fragment Analyzer system Agilent Santa Clara USA before sequencing.,R1272,SAMEA10486016,"Fraunhofer Attract Eco'n'OMICs, Fraunhofer Institute for Molecular Biology and Applied Ecology, Schmallenberg, Germany",ENA first public:2022 01 01|ENA last update:2022 01 01|External Id:SAMEA10486016|INSDC center alias:Fraunhofer Attract Eco'n'OMICs Fraunhofer Institute for Molecular Biology and Applied Ecology Schmallenberg Germany|INSDC center name:Fraunhofer Attract Eco'n'OMICs Fraunhofer Institute for Molecular Biology and Applied Ecology Schmallenberg Germany|INSDC first public:2022 01 01T00:13:13Z|INSDC last update:2022 01 01T00:13:13Z|INSDC status:public|Submitter Id:E MTAB 11092:R1272|age:51|broker name:ArrayExpress|common name:zebrafish|developmental stage:hatching long pec|genotype:wild type genotype|organism part:whole organism|replicate:T6|sample name:E MTAB 11092:R1272|sex:mixed|stimulus:water|strain:AB,,,,,,,,,Illumina HiSeq 4000 sequencing; Transcriptomic profiling of clobetasol propionate induced immunosuppression in challenged zebrafish embryos,E MTAB 11092:R1272 s,R1272 s,Transcriptomic profiling of clobetasol propionate induced immunosuppression in challenged zebrafish embryos,For each sample 10 zebrafish larvae were randomly picked from the glass well and carefully transferred to a 1.5 ml low binding Eppendorf tube. Once individuals were collected for all samples the supernatant was removed and the tubes were snap frozen in liquid nitrogen. For detailed information about embryonic incubation conditions and husbandry of adult broodstock please refer to the Growth protocol. For detailed information about exposure duration and conditions please refer to the Treatment protocol. Adult animal husbandry: Wild type zebrafish Danio rerio Strain AB broodstocks were maintained under flow through conditions in 150 L tanks at 26 +/ 2C on a 12:12 h light/dark cycle. They were fed daily with TetraMin Tetra Werke Melle Germany main feed ad libitum and nauplii of Artemia salina. The day before test start glass spawning trays with artificial substrate green glass beads stringed on stainless steel wire were placed at the bottom of each tank. post mating and spawning in the morning hours eggs were rinsed with clear Cu reduced water and placed into glass dishes for pre picking. Fish embryo incubation: For each sample 20 fertilized eggs in the early blastula stage were placed in glass petri dishes diameter 6 cm filled with 8 mL of the testing solution or control medium. Embryos were incubated at 27 +/ 1C on a 14:10 h light/dark cycle. At 24 hpf eggs were inspected while half of the test solution was renewed. Clobetasol propionate CAS: 25122 46 7 was purchased from Sigma Aldrich Taufkirchen Germany. For the test solution preparation first a 5 mM stock solution was prepared in Acetone 99.9% MS grade. This stock was diluted to 250 M in acetone before 500 L were added to a 500 mL DURAN glass bottle. To the bottle for the control solution only 500 L of pure Acetone was added. post complete evaporation of the acetone the bottle was filled up with 500 mL copper reduced tap water. Complete dissolution of precipitated clobetasol propionate was achieved by sonication for 30 minutes Sonorex RK 100H Bandelin Germany. The test solutions were freshly prepared one day prior the start of the experiment and were constantly aerated at room temperature. The glass wells for the incubation of the embryos were pre saturated with the respective test solution overnight and renewed at the beginning of the experiment. Per sample about 40 50 fertilized fish eggs were pre picked in glass petri dishes 10 cm diameter filled with 30 ml of the testing solution. Using an optical binokular microscope 20 embryos of the same early blastula stage were transferred from the pre picking plate into the test glass wells filled with 8 ml of the test solution. Embryos were then incubated as described in the growth protocol. Standard flagellin from Bacillus subtilis 10% purity Pam3CSK4 98 % purity and PolyI:C HMW high molecular weight were purchased from InvivoGen SAS Toulouse France. Stock solutions 1 mg/mL for polyI:C and Pam3CSK4; 0.1 mg/mL for flagellin were prepared in provided endotoxin free water aliquoted and stored according to the manufacturers instructions until further use. At approximately 46 hpf the embryos were manually dechorinated and then further incubated. A mixture of the different PAMPs was prepared by mixing 10 L of each component resulting in final concentrations of 0.33 mg/mL for polyI:C and Pam3CSK4 and 0.033 mg/mL for flagellin respectively. The device for microinjection was set up using FemtoTip II injection needles with a complementary universal capillary holder Eppendorf Hamburg Germany connected to an IM 300 microinjector Narishige London United Kingdom. Volume calibration was performed according to Sive et al. 2010 using a micrometer slide with 1/100 mm scale Bresser Rhede Germany. Briefly the needles were backfilled with 10 L PAMP mix or ultra pure water. Injections for volume calibration were made into a drop of immersion oil purred on the micrometer slide the output pressure was constantly kept at 10 psi while the time of injection was adjusted until an injected drop had a diameter of approximately 250 m. Assuming that the drop forms a perfect sphere this corresponds to a volume of about 8 nL. The procedure described below was performed for each of the five conditions in turn treating the three replicates simultaneously. In order to anaesthetize the embryos tricaine Sigma Aldrich Taufkirchen Germany stock solution 4 mg/mL in 21 mM Tris pH 9 was added to the petri dishes to a final concentration of 200 g/mL. The immobilized embryos were transferred onto a 1 % agarose plate which was divided into segments one for each replicate. Excess liquid was removed so that the embryos were kept in place by surface tension. Desiccation was prevented by adding liquid if needed. To ensure that the injections could be performed as quickly as possible the embryos were arranged in a row in a way that their dorsal sides pointed to the same direction. The puncture was made on the dorsal side of the yolk sac and the needle was advanced through the yolk until it reached the yolk sac circulation valley on the ventral side of the embryo where the injection was made directly into the bloodstream. post all replicates of a certain condition were processed the embryos were rinsed back into the corresponding dish containing renewed test solution. Total RNA was extracted using the NucleoSpin RNA/Protein kit from Macherey Nagel 740933.250 The pooled and snap frozen larvae were suspended in RP1 buffer and were transferred to a screw cap tubes filled with 0.3 g of Lysing Matrix D MP Biomedicals 6913050 ceramic beads. Tissue homogenization was performed at 5 m/s for 45 s with FastPrep 24 MP Biomedicals 6004500 at room temperature. From here on RNA was extracted according to the manufacturers protocol. RNA concentration >100 ng/l was measured using a Nanodrop 2000 spectrophotometer Thermo Scientific. The samples overall RNA quality and corresponding RIN value was fluorometrically determined with a 2100 Bioanalyzer Instrument system Agilent G2939BA using the Agilent RNA 6000 Nano Kit Agilent 5067 1511 according to the manufacturers protocol. Only samples with a RIN > 8 were used for downstream analysis. Samples were stored at 80C until they were send to the sequencing facility on dry ice. Sequencing libraries were prepared for each sample from 100 ng/l total RNA at the sequencing facility NGS Services for Integrative Genomics at the University of Gttingen in Germany. According to their standard workflow cDNA libraries were prepared from protein coding mRNA that were purified through PolyA selection using the TruSeq RNA Library Prep Kit v2 Illumina. Libraries were validated using a Fragment Analyzer system Agilent Santa Clara USA before sequencing.,Experimental Factor: compound:n1|Experimental Factor: stimulus:water,RNA-Seq,TRANSCRIPTOMIC,PolyA,SINGLE,ILLUMINA,Illumina HiSeq 4000,,ERP132743,Illumina HiSeq 4000 sequencing; Transcriptomic profiling of clobetasol propionate induced immunosuppression in challenged zebrafish embryos,ENA FIRST PUBLIC:2022 02 25|ENA LAST UPDATE:2022 02 25|loader:fastq load.py,p946sR1272_sr.fastq.gz,fastq,1634309317.0,32374943.0,E MTAB 11092:R1272,0:50.48,A:424752350;C:392450289;G:375596049;T:438931453;N:2579176,50,,,,424752350,392450289,375596049,438931453,2579176,ERX6749458,ERS8138764,ERA6860152,Fraunhofer Attract Eco,Fraunhofer Attract Eco,1,0.93744,,0.10119,,0.68627,,0.47078,,47,,B,,usable mapping rate,illumina,hiseq_era,unknown,poly_a,trueseq,bulk,unknown,unknown,,Germany,2022-01-01,Hatching,Embryo,Whole Organism,All anatomical structures
10313,ERR7179900,ERX6749457,ERS8138763,ERP132743,PRJEB48372,Transcriptomic profiling of clobetasol propionate induced immunosuppression in challenged zebrafish embryos,E-MTAB-11092,Transcriptome Analysis,The aim of this experiment was to develop an approach for the assessment of immunotoxicity in zebrafish embryos Danio rerio using transcriptomic profiling. Clobetasol propionate a highly potent synthetic corticosteroid which is known to have immunosuppressive effects on vertebrates including zebrafish was used for continuous exposure from fertilization until extraction. As immunotoxicity can only be comprehensively assessed when the effect is observed during an acute infection the embryos were injected with a mixture of different pathogen associated molecular patterns PAMPs consisting of Pam3CSK4 synthetic lipopeptide polyI:C synthetic viral dsRNA and flagellin isolated from Bacillus subtilis. Functional gene expression analysis from samples obtained by different kinds and combinations of treatments in relation to appropriate controls is used to identify pathways that are upregulated by artificial infection and suppressed by treatment with clobetasol propionate. In a modified version of the zebrafish embryo toxicity test OECD 236 20 fertilized eggs were exposed to a sub lethal concentration 250 nM of clobetasol propionate for xxx hours under semi static conditions. The test comprised of three treatments with clobetasol propionate and two negative control NC groups and was performed in triplicates. At 46 hpf the eggs were manually dechorinated. At 48 hpf the embryos were immobilized by anaesthetization using 200 mg/mL tricaine. Using microinjection 8 nL of the mixed PAMPs 0.33 µg/mL Pam3CSK4 0.33 µg/mL polyI:C and 0.033 µg/mL flagellin were injected directly into the embryos bloodstream. One of each exposure groups was injected with PAMPs while another one was injected with ultrapure water to control for effects introduced by the injection process. One control group remained completely untreated. post injection the embryos were further incubated for 3 hours before 10 embryos were randomly picked for each sample and pooled for RNA and protein extraction with NucleoSpin RNA/Protein kit Macherey Nagel. RNA quality was assessed with a 2100 Bioanalyzer system Agilent before coding RNA was purified PolyA selection with TruSeq RNA Library Prep Kit v2 and sequenced on an Illumina HiSeq 4000 System Illumina in 50 bp single read mode. Adapter sequences were removed with trimmomatic and sequences were aligned to the D.rerio reference genome GRCz11 with STAR. Counting of feature mapped reads was performed through featureCounts. Library gene count tables were then merged to a single count matrix as input for differential gene expression analysis with DESeq2.,ENA FIRST PUBLIC:2022 02 25|ENA LAST UPDATE:2022 02 25,,Protocols: For each sample 10 zebrafish larvae were randomly picked from the glass well and carefully transferred to a 1.5 ml low binding Eppendorf tube. Once individuals were collected for all samples the supernatant was removed and the tubes were snap frozen in liquid nitrogen. For detailed information about embryonic incubation conditions and husbandry of adult broodstock please refer to the “Growth protocol”. For detailed information about exposure duration and conditions please refer to the “Treatment protocol”. Adult animal husbandry: Wild type zebrafish Danio rerio Strain AB broodstocks were maintained under flow through conditions in 150 L tanks at 26 +/ 2°C on a 12:12 h light/dark cycle. They were fed daily with TetraMin® Tetra Werke Melle Germany main feed ad libitum and nauplii of Artemia salina. The day before test start glass spawning trays with artificial substrate green glass beads stringed on stainless steel wire were placed at the bottom of each tank. post mating and spawning in the morning hours eggs were rinsed with clear Cu reduced water and placed into glass dishes for pre picking. Fish embryo incubation: For each sample 20 fertilized eggs in the early blastula stage were placed in glass petri dishes diameter 6 cm filled with 8 mL of the testing solution or control medium. Embryos were incubated at 27 +/ 1°C on a 14:10 h light/dark cycle. At 24 hpf eggs were inspected while half of the test solution was renewed. Clobetasol propionate CAS: 25122 46 7 was purchased from Sigma Aldrich Taufkirchen Germany. For the test solution preparation first a 5 mM stock solution was prepared in Acetone 99.9% MS grade. This stock was diluted to 250 µM in acetone before 500 µL were added to a 500 mL DURAN glass bottle. To the bottle for the control solution only 500 µL of pure Acetone was added. post complete evaporation of the acetone the bottle was filled up with 500 mL copper reduced tap water. Complete dissolution of precipitated clobetasol propionate was achieved by sonication for 30 minutes Sonorex RK 100H Bandelin Germany. The test solutions were freshly prepared one day prior the start of the experiment and were constantly aerated at room temperature. The glass wells for the incubation of the embryos were pre saturated with the respective test solution overnight and renewed at the beginning of the experiment. Per sample about 40 50 fertilized fish eggs were pre picked in glass petri dishes 10 cm diameter filled with 30 ml of the testing solution. Using an optical binokular microscope 20 embryos of the same early blastula stage were transferred from the pre picking plate into the test glass wells filled with 8 ml of the test solution. Embryos were then incubated as described in the “growth protocol”. Standard flagellin from Bacillus subtilis ≈ 10% purity Pam3CSK4 ≥ 98 % purity and PolyI:C HMW high molecular weight were purchased from InvivoGen SAS Toulouse France. Stock solutions 1 mg/mL for polyI:C and Pam3CSK4; 0.1 mg/mL for flagellin were prepared in provided endotoxin free water aliquoted and stored according to the manufacturer's instructions until further use. At approximately 46 hpf the embryos were manually dechorinated and then further incubated. A mixture of the different PAMPs was prepared by mixing 10 µL of each component resulting in final concentrations of 0.33 mg/mL for polyI:C and Pam3CSK4 and 0.033 mg/mL for flagellin respectively. The device for microinjection was set up using FemtoTip II injection needles with a complementary universal capillary holder Eppendorf Hamburg Germany connected to an IM 300 microinjector Narishige London United Kingdom. Volume calibration was performed according to Sive et al. 2010 using a micrometer slide with 1/100 mm scale Bresser Rhede Germany. Briefly the needles were backfilled with 10 µL PAMP mix or ultra pure water. Injections for volume calibration were made into a drop of immersion oil purred on the micrometer slide the output pressure was constantly kept at 10 psi while the time of injection was adjusted until an injected drop had a diameter of approximately 250 µm. Assuming that the drop forms a perfect sphere this corresponds to a volume of about 8 nL. The procedure described below was performed for each of the five conditions in turn treating the three replicates simultaneously. In order to anaesthetize the embryos tricaine Sigma Aldrich Taufkirchen Germany stock solution 4 mg/mL in 21 mM Tris pH 9 was added to the petri dishes to a final concentration of 200 µg/mL. The immobilized embryos were transferred onto a 1 % agarose plate which was divided into segments one for each replicate. Excess liquid was removed so that the embryos were kept in place by surface tension. Desiccation was prevented by adding liquid if needed. To ensure that the injections could be performed as quickly as possible the embryos were arranged in a row in a way that their dorsal sides pointed to the same direction. The puncture was made on the dorsal side of the yolk sac and the needle was advanced through the yolk until it reached the yolk sac circulation valley on the ventral side of the embryo where the injection was made directly into the bloodstream. post all replicates of a certain condition were processed the embryos were rinsed back into the corresponding dish containing renewed test solution. Total RNA was extracted using the NucleoSpin RNA/Protein kit from Macherey Nagel 740933.250 The pooled and snap frozen larvae were suspended in RP1 buffer and were transferred to a screw cap tubes filled with 0.3 g of Lysing Matrix D MP Biomedicals 6913050 ceramic beads. Tissue homogenization was performed at 5 m/s for 45 s with FastPrep 24© MP Biomedicals 6004500 at room temperature. From here on RNA was extracted according to the manufacturer's protocol. RNA concentration >100 ng/µl was measured using a Nanodrop 2000 spectrophotometer Thermo Scientific. The sample's overall RNA quality and corresponding RIN value was fluorometrically determined with a 2100 Bioanalyzer© Instrument system Agilent G2939BA using the Agilent RNA 6000 Nano Kit© Agilent 5067 1511 according to the manufacturer's protocol. Only samples with a RIN > 8 were used for downstream analysis. Samples were stored at 80°C until they were send to the sequencing facility on dry ice. Sequencing libraries were prepared for each sample from 100 ng/µl total RNA at the sequencing facility “NGS Services for Integrative Genomics” at the University of Göttingen in Germany. According to their standard workflow cDNA libraries were prepared from protein coding mRNA that were purified through PolyA selection using the TruSeq RNA Library Prep Kit v2 Illumina. Libraries were validated using a Fragment Analyzer system Agilent Santa Clara USA before sequencing.,R1269,SAMEA10486014,"Fraunhofer Attract Eco'n'OMICs, Fraunhofer Institute for Molecular Biology and Applied Ecology, Schmallenberg, Germany",ENA first public:2022 01 01|ENA last update:2022 01 01|External Id:SAMEA10486014|INSDC center alias:Fraunhofer Attract Eco'n'OMICs Fraunhofer Institute for Molecular Biology and Applied Ecology Schmallenberg Germany|INSDC center name:Fraunhofer Attract Eco'n'OMICs Fraunhofer Institute for Molecular Biology and Applied Ecology Schmallenberg Germany|INSDC first public:2022 01 01T00:13:13Z|INSDC last update:2022 01 01T00:13:13Z|INSDC status:public|Submitter Id:E MTAB 11092:R1269|age:51|broker name:ArrayExpress|common name:zebrafish|developmental stage:hatching long pec|genotype:wild type genotype|organism part:whole organism|replicate:T6|sample name:E MTAB 11092:R1269|sex:mixed|stimulus:control|strain:AB,,,,,,,,,Illumina HiSeq 4000 sequencing; Transcriptomic profiling of clobetasol propionate induced immunosuppression in challenged zebrafish embryos,E MTAB 11092:R1269 s,R1269 s,Transcriptomic profiling of clobetasol propionate induced immunosuppression in challenged zebrafish embryos,For each sample 10 zebrafish larvae were randomly picked from the glass well and carefully transferred to a 1.5 ml low binding Eppendorf tube. Once individuals were collected for all samples the supernatant was removed and the tubes were snap frozen in liquid nitrogen. For detailed information about embryonic incubation conditions and husbandry of adult broodstock please refer to the Growth protocol. For detailed information about exposure duration and conditions please refer to the Treatment protocol. Adult animal husbandry: Wild type zebrafish Danio rerio Strain AB broodstocks were maintained under flow through conditions in 150 L tanks at 26 +/ 2C on a 12:12 h light/dark cycle. They were fed daily with TetraMin Tetra Werke Melle Germany main feed ad libitum and nauplii of Artemia salina. The day before test start glass spawning trays with artificial substrate green glass beads stringed on stainless steel wire were placed at the bottom of each tank. post mating and spawning in the morning hours eggs were rinsed with clear Cu reduced water and placed into glass dishes for pre picking. Fish embryo incubation: For each sample 20 fertilized eggs in the early blastula stage were placed in glass petri dishes diameter 6 cm filled with 8 mL of the testing solution or control medium. Embryos were incubated at 27 +/ 1C on a 14:10 h light/dark cycle. At 24 hpf eggs were inspected while half of the test solution was renewed. Clobetasol propionate CAS: 25122 46 7 was purchased from Sigma Aldrich Taufkirchen Germany. For the test solution preparation first a 5 mM stock solution was prepared in Acetone 99.9% MS grade. This stock was diluted to 250 M in acetone before 500 L were added to a 500 mL DURAN glass bottle. To the bottle for the control solution only 500 L of pure Acetone was added. post complete evaporation of the acetone the bottle was filled up with 500 mL copper reduced tap water. Complete dissolution of precipitated clobetasol propionate was achieved by sonication for 30 minutes Sonorex RK 100H Bandelin Germany. The test solutions were freshly prepared one day prior the start of the experiment and were constantly aerated at room temperature. The glass wells for the incubation of the embryos were pre saturated with the respective test solution overnight and renewed at the beginning of the experiment. Per sample about 40 50 fertilized fish eggs were pre picked in glass petri dishes 10 cm diameter filled with 30 ml of the testing solution. Using an optical binokular microscope 20 embryos of the same early blastula stage were transferred from the pre picking plate into the test glass wells filled with 8 ml of the test solution. Embryos were then incubated as described in the growth protocol. Standard flagellin from Bacillus subtilis 10% purity Pam3CSK4 98 % purity and PolyI:C HMW high molecular weight were purchased from InvivoGen SAS Toulouse France. Stock solutions 1 mg/mL for polyI:C and Pam3CSK4; 0.1 mg/mL for flagellin were prepared in provided endotoxin free water aliquoted and stored according to the manufacturers instructions until further use. At approximately 46 hpf the embryos were manually dechorinated and then further incubated. A mixture of the different PAMPs was prepared by mixing 10 L of each component resulting in final concentrations of 0.33 mg/mL for polyI:C and Pam3CSK4 and 0.033 mg/mL for flagellin respectively. The device for microinjection was set up using FemtoTip II injection needles with a complementary universal capillary holder Eppendorf Hamburg Germany connected to an IM 300 microinjector Narishige London United Kingdom. Volume calibration was performed according to Sive et al. 2010 using a micrometer slide with 1/100 mm scale Bresser Rhede Germany. Briefly the needles were backfilled with 10 L PAMP mix or ultra pure water. Injections for volume calibration were made into a drop of immersion oil purred on the micrometer slide the output pressure was constantly kept at 10 psi while the time of injection was adjusted until an injected drop had a diameter of approximately 250 m. Assuming that the drop forms a perfect sphere this corresponds to a volume of about 8 nL. The procedure described below was performed for each of the five conditions in turn treating the three replicates simultaneously. In order to anaesthetize the embryos tricaine Sigma Aldrich Taufkirchen Germany stock solution 4 mg/mL in 21 mM Tris pH 9 was added to the petri dishes to a final concentration of 200 g/mL. The immobilized embryos were transferred onto a 1 % agarose plate which was divided into segments one for each replicate. Excess liquid was removed so that the embryos were kept in place by surface tension. Desiccation was prevented by adding liquid if needed. To ensure that the injections could be performed as quickly as possible the embryos were arranged in a row in a way that their dorsal sides pointed to the same direction. The puncture was made on the dorsal side of the yolk sac and the needle was advanced through the yolk until it reached the yolk sac circulation valley on the ventral side of the embryo where the injection was made directly into the bloodstream. post all replicates of a certain condition were processed the embryos were rinsed back into the corresponding dish containing renewed test solution. Total RNA was extracted using the NucleoSpin RNA/Protein kit from Macherey Nagel 740933.250 The pooled and snap frozen larvae were suspended in RP1 buffer and were transferred to a screw cap tubes filled with 0.3 g of Lysing Matrix D MP Biomedicals 6913050 ceramic beads. Tissue homogenization was performed at 5 m/s for 45 s with FastPrep 24 MP Biomedicals 6004500 at room temperature. From here on RNA was extracted according to the manufacturers protocol. RNA concentration >100 ng/l was measured using a Nanodrop 2000 spectrophotometer Thermo Scientific. The samples overall RNA quality and corresponding RIN value was fluorometrically determined with a 2100 Bioanalyzer Instrument system Agilent G2939BA using the Agilent RNA 6000 Nano Kit Agilent 5067 1511 according to the manufacturers protocol. Only samples with a RIN > 8 were used for downstream analysis. Samples were stored at 80C until they were send to the sequencing facility on dry ice. Sequencing libraries were prepared for each sample from 100 ng/l total RNA at the sequencing facility NGS Services for Integrative Genomics at the University of Gttingen in Germany. According to their standard workflow cDNA libraries were prepared from protein coding mRNA that were purified through PolyA selection using the TruSeq RNA Library Prep Kit v2 Illumina. Libraries were validated using a Fragment Analyzer system Agilent Santa Clara USA before sequencing.,Experimental Factor: compound:n1|Experimental Factor: stimulus:n1,RNA-Seq,TRANSCRIPTOMIC,PolyA,SINGLE,ILLUMINA,Illumina HiSeq 4000,,ERP132743,Illumina HiSeq 4000 sequencing; Transcriptomic profiling of clobetasol propionate induced immunosuppression in challenged zebrafish embryos,ENA FIRST PUBLIC:2022 02 25|ENA LAST UPDATE:2022 02 25|loader:fastq load.py,p946sR1269_sr.fastq.gz,fastq,1209767720.0,23959130.0,E MTAB 11092:R1269,0:50.49,A:311532826;C:293995213;G:280433261;T:322310486;N:1495934,50,,,,311532826,293995213,280433261,322310486,1495934,ERX6749457,ERS8138763,ERA6860152,Fraunhofer Attract Eco,Fraunhofer Attract Eco,1,0.94226,,0.10041,,0.68777,,0.45677,,50,,B,,usable mapping rate,illumina,hiseq_era,unknown,poly_a,trueseq,bulk,unknown,unknown,,Germany,2022-01-01,Hatching,Embryo,Whole Organism,All anatomical structures
10314,ERR7179899,ERX6749456,ERS8138762,ERP132743,PRJEB48372,Transcriptomic profiling of clobetasol propionate induced immunosuppression in challenged zebrafish embryos,E-MTAB-11092,Transcriptome Analysis,The aim of this experiment was to develop an approach for the assessment of immunotoxicity in zebrafish embryos Danio rerio using transcriptomic profiling. Clobetasol propionate a highly potent synthetic corticosteroid which is known to have immunosuppressive effects on vertebrates including zebrafish was used for continuous exposure from fertilization until extraction. As immunotoxicity can only be comprehensively assessed when the effect is observed during an acute infection the embryos were injected with a mixture of different pathogen associated molecular patterns PAMPs consisting of Pam3CSK4 synthetic lipopeptide polyI:C synthetic viral dsRNA and flagellin isolated from Bacillus subtilis. Functional gene expression analysis from samples obtained by different kinds and combinations of treatments in relation to appropriate controls is used to identify pathways that are upregulated by artificial infection and suppressed by treatment with clobetasol propionate. In a modified version of the zebrafish embryo toxicity test OECD 236 20 fertilized eggs were exposed to a sub lethal concentration 250 nM of clobetasol propionate for xxx hours under semi static conditions. The test comprised of three treatments with clobetasol propionate and two negative control NC groups and was performed in triplicates. At 46 hpf the eggs were manually dechorinated. At 48 hpf the embryos were immobilized by anaesthetization using 200 mg/mL tricaine. Using microinjection 8 nL of the mixed PAMPs 0.33 µg/mL Pam3CSK4 0.33 µg/mL polyI:C and 0.033 µg/mL flagellin were injected directly into the embryos bloodstream. One of each exposure groups was injected with PAMPs while another one was injected with ultrapure water to control for effects introduced by the injection process. One control group remained completely untreated. post injection the embryos were further incubated for 3 hours before 10 embryos were randomly picked for each sample and pooled for RNA and protein extraction with NucleoSpin RNA/Protein kit Macherey Nagel. RNA quality was assessed with a 2100 Bioanalyzer system Agilent before coding RNA was purified PolyA selection with TruSeq RNA Library Prep Kit v2 and sequenced on an Illumina HiSeq 4000 System Illumina in 50 bp single read mode. Adapter sequences were removed with trimmomatic and sequences were aligned to the D.rerio reference genome GRCz11 with STAR. Counting of feature mapped reads was performed through featureCounts. Library gene count tables were then merged to a single count matrix as input for differential gene expression analysis with DESeq2.,ENA FIRST PUBLIC:2022 02 25|ENA LAST UPDATE:2022 02 25,,Protocols: For each sample 10 zebrafish larvae were randomly picked from the glass well and carefully transferred to a 1.5 ml low binding Eppendorf tube. Once individuals were collected for all samples the supernatant was removed and the tubes were snap frozen in liquid nitrogen. For detailed information about embryonic incubation conditions and husbandry of adult broodstock please refer to the “Growth protocol”. For detailed information about exposure duration and conditions please refer to the “Treatment protocol”. Adult animal husbandry: Wild type zebrafish Danio rerio Strain AB broodstocks were maintained under flow through conditions in 150 L tanks at 26 +/ 2°C on a 12:12 h light/dark cycle. They were fed daily with TetraMin® Tetra Werke Melle Germany main feed ad libitum and nauplii of Artemia salina. The day before test start glass spawning trays with artificial substrate green glass beads stringed on stainless steel wire were placed at the bottom of each tank. post mating and spawning in the morning hours eggs were rinsed with clear Cu reduced water and placed into glass dishes for pre picking. Fish embryo incubation: For each sample 20 fertilized eggs in the early blastula stage were placed in glass petri dishes diameter 6 cm filled with 8 mL of the testing solution or control medium. Embryos were incubated at 27 +/ 1°C on a 14:10 h light/dark cycle. At 24 hpf eggs were inspected while half of the test solution was renewed. Clobetasol propionate CAS: 25122 46 7 was purchased from Sigma Aldrich Taufkirchen Germany. For the test solution preparation first a 5 mM stock solution was prepared in Acetone 99.9% MS grade. This stock was diluted to 250 µM in acetone before 500 µL were added to a 500 mL DURAN glass bottle. To the bottle for the control solution only 500 µL of pure Acetone was added. post complete evaporation of the acetone the bottle was filled up with 500 mL copper reduced tap water. Complete dissolution of precipitated clobetasol propionate was achieved by sonication for 30 minutes Sonorex RK 100H Bandelin Germany. The test solutions were freshly prepared one day prior the start of the experiment and were constantly aerated at room temperature. The glass wells for the incubation of the embryos were pre saturated with the respective test solution overnight and renewed at the beginning of the experiment. Per sample about 40 50 fertilized fish eggs were pre picked in glass petri dishes 10 cm diameter filled with 30 ml of the testing solution. Using an optical binokular microscope 20 embryos of the same early blastula stage were transferred from the pre picking plate into the test glass wells filled with 8 ml of the test solution. Embryos were then incubated as described in the “growth protocol”. Standard flagellin from Bacillus subtilis ≈ 10% purity Pam3CSK4 ≥ 98 % purity and PolyI:C HMW high molecular weight were purchased from InvivoGen SAS Toulouse France. Stock solutions 1 mg/mL for polyI:C and Pam3CSK4; 0.1 mg/mL for flagellin were prepared in provided endotoxin free water aliquoted and stored according to the manufacturer's instructions until further use. At approximately 46 hpf the embryos were manually dechorinated and then further incubated. A mixture of the different PAMPs was prepared by mixing 10 µL of each component resulting in final concentrations of 0.33 mg/mL for polyI:C and Pam3CSK4 and 0.033 mg/mL for flagellin respectively. The device for microinjection was set up using FemtoTip II injection needles with a complementary universal capillary holder Eppendorf Hamburg Germany connected to an IM 300 microinjector Narishige London United Kingdom. Volume calibration was performed according to Sive et al. 2010 using a micrometer slide with 1/100 mm scale Bresser Rhede Germany. Briefly the needles were backfilled with 10 µL PAMP mix or ultra pure water. Injections for volume calibration were made into a drop of immersion oil purred on the micrometer slide the output pressure was constantly kept at 10 psi while the time of injection was adjusted until an injected drop had a diameter of approximately 250 µm. Assuming that the drop forms a perfect sphere this corresponds to a volume of about 8 nL. The procedure described below was performed for each of the five conditions in turn treating the three replicates simultaneously. In order to anaesthetize the embryos tricaine Sigma Aldrich Taufkirchen Germany stock solution 4 mg/mL in 21 mM Tris pH 9 was added to the petri dishes to a final concentration of 200 µg/mL. The immobilized embryos were transferred onto a 1 % agarose plate which was divided into segments one for each replicate. Excess liquid was removed so that the embryos were kept in place by surface tension. Desiccation was prevented by adding liquid if needed. To ensure that the injections could be performed as quickly as possible the embryos were arranged in a row in a way that their dorsal sides pointed to the same direction. The puncture was made on the dorsal side of the yolk sac and the needle was advanced through the yolk until it reached the yolk sac circulation valley on the ventral side of the embryo where the injection was made directly into the bloodstream. post all replicates of a certain condition were processed the embryos were rinsed back into the corresponding dish containing renewed test solution. Total RNA was extracted using the NucleoSpin RNA/Protein kit from Macherey Nagel 740933.250 The pooled and snap frozen larvae were suspended in RP1 buffer and were transferred to a screw cap tubes filled with 0.3 g of Lysing Matrix D MP Biomedicals 6913050 ceramic beads. Tissue homogenization was performed at 5 m/s for 45 s with FastPrep 24© MP Biomedicals 6004500 at room temperature. From here on RNA was extracted according to the manufacturer's protocol. RNA concentration >100 ng/µl was measured using a Nanodrop 2000 spectrophotometer Thermo Scientific. The sample's overall RNA quality and corresponding RIN value was fluorometrically determined with a 2100 Bioanalyzer© Instrument system Agilent G2939BA using the Agilent RNA 6000 Nano Kit© Agilent 5067 1511 according to the manufacturer's protocol. Only samples with a RIN > 8 were used for downstream analysis. Samples were stored at 80°C until they were send to the sequencing facility on dry ice. Sequencing libraries were prepared for each sample from 100 ng/µl total RNA at the sequencing facility “NGS Services for Integrative Genomics” at the University of Göttingen in Germany. According to their standard workflow cDNA libraries were prepared from protein coding mRNA that were purified through PolyA selection using the TruSeq RNA Library Prep Kit v2 Illumina. Libraries were validated using a Fragment Analyzer system Agilent Santa Clara USA before sequencing.,R1289,SAMEA10486012,"Fraunhofer Attract Eco'n'OMICs, Fraunhofer Institute for Molecular Biology and Applied Ecology, Schmallenberg, Germany",ENA first public:2022 01 01|ENA last update:2022 01 01|External Id:SAMEA10486012|INSDC center alias:Fraunhofer Attract Eco'n'OMICs Fraunhofer Institute for Molecular Biology and Applied Ecology Schmallenberg Germany|INSDC center name:Fraunhofer Attract Eco'n'OMICs Fraunhofer Institute for Molecular Biology and Applied Ecology Schmallenberg Germany|INSDC first public:2022 01 01T00:13:13Z|INSDC last update:2022 01 01T00:13:13Z|INSDC status:public|Submitter Id:E MTAB 11092:R1289|age:51|broker name:ArrayExpress|common name:zebrafish|developmental stage:hatching long pec|genotype:wild type genotype|organism part:whole organism|replicate:T13|sample name:E MTAB 11092:R1289|sex:mixed|stimulus:PAMP|strain:AB,,,,,,,,,Illumina HiSeq 4000 sequencing; Transcriptomic profiling of clobetasol propionate induced immunosuppression in challenged zebrafish embryos,E MTAB 11092:R1289 s,R1289 s,Transcriptomic profiling of clobetasol propionate induced immunosuppression in challenged zebrafish embryos,For each sample 10 zebrafish larvae were randomly picked from the glass well and carefully transferred to a 1.5 ml low binding Eppendorf tube. Once individuals were collected for all samples the supernatant was removed and the tubes were snap frozen in liquid nitrogen. For detailed information about embryonic incubation conditions and husbandry of adult broodstock please refer to the Growth protocol. For detailed information about exposure duration and conditions please refer to the Treatment protocol. Adult animal husbandry: Wild type zebrafish Danio rerio Strain AB broodstocks were maintained under flow through conditions in 150 L tanks at 26 +/ 2C on a 12:12 h light/dark cycle. They were fed daily with TetraMin Tetra Werke Melle Germany main feed ad libitum and nauplii of Artemia salina. The day before test start glass spawning trays with artificial substrate green glass beads stringed on stainless steel wire were placed at the bottom of each tank. post mating and spawning in the morning hours eggs were rinsed with clear Cu reduced water and placed into glass dishes for pre picking. Fish embryo incubation: For each sample 20 fertilized eggs in the early blastula stage were placed in glass petri dishes diameter 6 cm filled with 8 mL of the testing solution or control medium. Embryos were incubated at 27 +/ 1C on a 14:10 h light/dark cycle. At 24 hpf eggs were inspected while half of the test solution was renewed. Clobetasol propionate CAS: 25122 46 7 was purchased from Sigma Aldrich Taufkirchen Germany. For the test solution preparation first a 5 mM stock solution was prepared in Acetone 99.9% MS grade. This stock was diluted to 250 M in acetone before 500 L were added to a 500 mL DURAN glass bottle. To the bottle for the control solution only 500 L of pure Acetone was added. post complete evaporation of the acetone the bottle was filled up with 500 mL copper reduced tap water. Complete dissolution of precipitated clobetasol propionate was achieved by sonication for 30 minutes Sonorex RK 100H Bandelin Germany. The test solutions were freshly prepared one day prior the start of the experiment and were constantly aerated at room temperature. The glass wells for the incubation of the embryos were pre saturated with the respective test solution overnight and renewed at the beginning of the experiment. Per sample about 40 50 fertilized fish eggs were pre picked in glass petri dishes 10 cm diameter filled with 30 ml of the testing solution. Using an optical binokular microscope 20 embryos of the same early blastula stage were transferred from the pre picking plate into the test glass wells filled with 8 ml of the test solution. Embryos were then incubated as described in the growth protocol. Standard flagellin from Bacillus subtilis 10% purity Pam3CSK4 98 % purity and PolyI:C HMW high molecular weight were purchased from InvivoGen SAS Toulouse France. Stock solutions 1 mg/mL for polyI:C and Pam3CSK4; 0.1 mg/mL for flagellin were prepared in provided endotoxin free water aliquoted and stored according to the manufacturers instructions until further use. At approximately 46 hpf the embryos were manually dechorinated and then further incubated. A mixture of the different PAMPs was prepared by mixing 10 L of each component resulting in final concentrations of 0.33 mg/mL for polyI:C and Pam3CSK4 and 0.033 mg/mL for flagellin respectively. The device for microinjection was set up using FemtoTip II injection needles with a complementary universal capillary holder Eppendorf Hamburg Germany connected to an IM 300 microinjector Narishige London United Kingdom. Volume calibration was performed according to Sive et al. 2010 using a micrometer slide with 1/100 mm scale Bresser Rhede Germany. Briefly the needles were backfilled with 10 L PAMP mix or ultra pure water. Injections for volume calibration were made into a drop of immersion oil purred on the micrometer slide the output pressure was constantly kept at 10 psi while the time of injection was adjusted until an injected drop had a diameter of approximately 250 m. Assuming that the drop forms a perfect sphere this corresponds to a volume of about 8 nL. The procedure described below was performed for each of the five conditions in turn treating the three replicates simultaneously. In order to anaesthetize the embryos tricaine Sigma Aldrich Taufkirchen Germany stock solution 4 mg/mL in 21 mM Tris pH 9 was added to the petri dishes to a final concentration of 200 g/mL. The immobilized embryos were transferred onto a 1 % agarose plate which was divided into segments one for each replicate. Excess liquid was removed so that the embryos were kept in place by surface tension. Desiccation was prevented by adding liquid if needed. To ensure that the injections could be performed as quickly as possible the embryos were arranged in a row in a way that their dorsal sides pointed to the same direction. The puncture was made on the dorsal side of the yolk sac and the needle was advanced through the yolk until it reached the yolk sac circulation valley on the ventral side of the embryo where the injection was made directly into the bloodstream. post all replicates of a certain condition were processed the embryos were rinsed back into the corresponding dish containing renewed test solution. Total RNA was extracted using the NucleoSpin RNA/Protein kit from Macherey Nagel 740933.250 The pooled and snap frozen larvae were suspended in RP1 buffer and were transferred to a screw cap tubes filled with 0.3 g of Lysing Matrix D MP Biomedicals 6913050 ceramic beads. Tissue homogenization was performed at 5 m/s for 45 s with FastPrep 24 MP Biomedicals 6004500 at room temperature. From here on RNA was extracted according to the manufacturers protocol. RNA concentration >100 ng/l was measured using a Nanodrop 2000 spectrophotometer Thermo Scientific. The samples overall RNA quality and corresponding RIN value was fluorometrically determined with a 2100 Bioanalyzer Instrument system Agilent G2939BA using the Agilent RNA 6000 Nano Kit Agilent 5067 1511 according to the manufacturers protocol. Only samples with a RIN > 8 were used for downstream analysis. Samples were stored at 80C until they were send to the sequencing facility on dry ice. Sequencing libraries were prepared for each sample from 100 ng/l total RNA at the sequencing facility NGS Services for Integrative Genomics at the University of Gttingen in Germany. According to their standard workflow cDNA libraries were prepared from protein coding mRNA that were purified through PolyA selection using the TruSeq RNA Library Prep Kit v2 Illumina. Libraries were validated using a Fragment Analyzer system Agilent Santa Clara USA before sequencing.,Experimental Factor: compound:Clobetasol propionate|Experimental Factor: dose:250|Experimental Factor: stimulus:PAMP,RNA-Seq,TRANSCRIPTOMIC,PolyA,SINGLE,ILLUMINA,Illumina HiSeq 4000,,ERP132743,Illumina HiSeq 4000 sequencing; Transcriptomic profiling of clobetasol propionate induced immunosuppression in challenged zebrafish embryos,ENA FIRST PUBLIC:2022 02 25|ENA LAST UPDATE:2022 02 25|loader:fastq load.py,p946sR1289_sr.fastq.gz,fastq,2336898428.0,46306897.0,E MTAB 11092:R1289,0:50.47,A:612494442;C:551754242;G:532830428;T:633879257;N:5940059,50,,,,612494442,551754242,532830428,633879257,5940059,ERX6749456,ERS8138762,ERA6860152,Fraunhofer Attract Eco,Fraunhofer Attract Eco,1,0.93744,,0.13096,,0.68753,,0.49027,,50,,B,,usable mapping rate,illumina,hiseq_era,unknown,poly_a,trueseq,bulk,unknown,unknown,,Germany,2022-01-01,Hatching,Embryo,Whole Organism,All anatomical structures
10315,ERR7179898,ERX6749455,ERS8138761,ERP132743,PRJEB48372,Transcriptomic profiling of clobetasol propionate induced immunosuppression in challenged zebrafish embryos,E-MTAB-11092,Transcriptome Analysis,The aim of this experiment was to develop an approach for the assessment of immunotoxicity in zebrafish embryos Danio rerio using transcriptomic profiling. Clobetasol propionate a highly potent synthetic corticosteroid which is known to have immunosuppressive effects on vertebrates including zebrafish was used for continuous exposure from fertilization until extraction. As immunotoxicity can only be comprehensively assessed when the effect is observed during an acute infection the embryos were injected with a mixture of different pathogen associated molecular patterns PAMPs consisting of Pam3CSK4 synthetic lipopeptide polyI:C synthetic viral dsRNA and flagellin isolated from Bacillus subtilis. Functional gene expression analysis from samples obtained by different kinds and combinations of treatments in relation to appropriate controls is used to identify pathways that are upregulated by artificial infection and suppressed by treatment with clobetasol propionate. In a modified version of the zebrafish embryo toxicity test OECD 236 20 fertilized eggs were exposed to a sub lethal concentration 250 nM of clobetasol propionate for xxx hours under semi static conditions. The test comprised of three treatments with clobetasol propionate and two negative control NC groups and was performed in triplicates. At 46 hpf the eggs were manually dechorinated. At 48 hpf the embryos were immobilized by anaesthetization using 200 mg/mL tricaine. Using microinjection 8 nL of the mixed PAMPs 0.33 µg/mL Pam3CSK4 0.33 µg/mL polyI:C and 0.033 µg/mL flagellin were injected directly into the embryos bloodstream. One of each exposure groups was injected with PAMPs while another one was injected with ultrapure water to control for effects introduced by the injection process. One control group remained completely untreated. post injection the embryos were further incubated for 3 hours before 10 embryos were randomly picked for each sample and pooled for RNA and protein extraction with NucleoSpin RNA/Protein kit Macherey Nagel. RNA quality was assessed with a 2100 Bioanalyzer system Agilent before coding RNA was purified PolyA selection with TruSeq RNA Library Prep Kit v2 and sequenced on an Illumina HiSeq 4000 System Illumina in 50 bp single read mode. Adapter sequences were removed with trimmomatic and sequences were aligned to the D.rerio reference genome GRCz11 with STAR. Counting of feature mapped reads was performed through featureCounts. Library gene count tables were then merged to a single count matrix as input for differential gene expression analysis with DESeq2.,ENA FIRST PUBLIC:2022 02 25|ENA LAST UPDATE:2022 02 25,,Protocols: For each sample 10 zebrafish larvae were randomly picked from the glass well and carefully transferred to a 1.5 ml low binding Eppendorf tube. Once individuals were collected for all samples the supernatant was removed and the tubes were snap frozen in liquid nitrogen. For detailed information about embryonic incubation conditions and husbandry of adult broodstock please refer to the “Growth protocol”. For detailed information about exposure duration and conditions please refer to the “Treatment protocol”. Adult animal husbandry: Wild type zebrafish Danio rerio Strain AB broodstocks were maintained under flow through conditions in 150 L tanks at 26 +/ 2°C on a 12:12 h light/dark cycle. They were fed daily with TetraMin® Tetra Werke Melle Germany main feed ad libitum and nauplii of Artemia salina. The day before test start glass spawning trays with artificial substrate green glass beads stringed on stainless steel wire were placed at the bottom of each tank. post mating and spawning in the morning hours eggs were rinsed with clear Cu reduced water and placed into glass dishes for pre picking. Fish embryo incubation: For each sample 20 fertilized eggs in the early blastula stage were placed in glass petri dishes diameter 6 cm filled with 8 mL of the testing solution or control medium. Embryos were incubated at 27 +/ 1°C on a 14:10 h light/dark cycle. At 24 hpf eggs were inspected while half of the test solution was renewed. Clobetasol propionate CAS: 25122 46 7 was purchased from Sigma Aldrich Taufkirchen Germany. For the test solution preparation first a 5 mM stock solution was prepared in Acetone 99.9% MS grade. This stock was diluted to 250 µM in acetone before 500 µL were added to a 500 mL DURAN glass bottle. To the bottle for the control solution only 500 µL of pure Acetone was added. post complete evaporation of the acetone the bottle was filled up with 500 mL copper reduced tap water. Complete dissolution of precipitated clobetasol propionate was achieved by sonication for 30 minutes Sonorex RK 100H Bandelin Germany. The test solutions were freshly prepared one day prior the start of the experiment and were constantly aerated at room temperature. The glass wells for the incubation of the embryos were pre saturated with the respective test solution overnight and renewed at the beginning of the experiment. Per sample about 40 50 fertilized fish eggs were pre picked in glass petri dishes 10 cm diameter filled with 30 ml of the testing solution. Using an optical binokular microscope 20 embryos of the same early blastula stage were transferred from the pre picking plate into the test glass wells filled with 8 ml of the test solution. Embryos were then incubated as described in the “growth protocol”. Standard flagellin from Bacillus subtilis ≈ 10% purity Pam3CSK4 ≥ 98 % purity and PolyI:C HMW high molecular weight were purchased from InvivoGen SAS Toulouse France. Stock solutions 1 mg/mL for polyI:C and Pam3CSK4; 0.1 mg/mL for flagellin were prepared in provided endotoxin free water aliquoted and stored according to the manufacturer's instructions until further use. At approximately 46 hpf the embryos were manually dechorinated and then further incubated. A mixture of the different PAMPs was prepared by mixing 10 µL of each component resulting in final concentrations of 0.33 mg/mL for polyI:C and Pam3CSK4 and 0.033 mg/mL for flagellin respectively. The device for microinjection was set up using FemtoTip II injection needles with a complementary universal capillary holder Eppendorf Hamburg Germany connected to an IM 300 microinjector Narishige London United Kingdom. Volume calibration was performed according to Sive et al. 2010 using a micrometer slide with 1/100 mm scale Bresser Rhede Germany. Briefly the needles were backfilled with 10 µL PAMP mix or ultra pure water. Injections for volume calibration were made into a drop of immersion oil purred on the micrometer slide the output pressure was constantly kept at 10 psi while the time of injection was adjusted until an injected drop had a diameter of approximately 250 µm. Assuming that the drop forms a perfect sphere this corresponds to a volume of about 8 nL. The procedure described below was performed for each of the five conditions in turn treating the three replicates simultaneously. In order to anaesthetize the embryos tricaine Sigma Aldrich Taufkirchen Germany stock solution 4 mg/mL in 21 mM Tris pH 9 was added to the petri dishes to a final concentration of 200 µg/mL. The immobilized embryos were transferred onto a 1 % agarose plate which was divided into segments one for each replicate. Excess liquid was removed so that the embryos were kept in place by surface tension. Desiccation was prevented by adding liquid if needed. To ensure that the injections could be performed as quickly as possible the embryos were arranged in a row in a way that their dorsal sides pointed to the same direction. The puncture was made on the dorsal side of the yolk sac and the needle was advanced through the yolk until it reached the yolk sac circulation valley on the ventral side of the embryo where the injection was made directly into the bloodstream. post all replicates of a certain condition were processed the embryos were rinsed back into the corresponding dish containing renewed test solution. Total RNA was extracted using the NucleoSpin RNA/Protein kit from Macherey Nagel 740933.250 The pooled and snap frozen larvae were suspended in RP1 buffer and were transferred to a screw cap tubes filled with 0.3 g of Lysing Matrix D MP Biomedicals 6913050 ceramic beads. Tissue homogenization was performed at 5 m/s for 45 s with FastPrep 24© MP Biomedicals 6004500 at room temperature. From here on RNA was extracted according to the manufacturer's protocol. RNA concentration >100 ng/µl was measured using a Nanodrop 2000 spectrophotometer Thermo Scientific. The sample's overall RNA quality and corresponding RIN value was fluorometrically determined with a 2100 Bioanalyzer© Instrument system Agilent G2939BA using the Agilent RNA 6000 Nano Kit© Agilent 5067 1511 according to the manufacturer's protocol. Only samples with a RIN > 8 were used for downstream analysis. Samples were stored at 80°C until they were send to the sequencing facility on dry ice. Sequencing libraries were prepared for each sample from 100 ng/µl total RNA at the sequencing facility “NGS Services for Integrative Genomics” at the University of Göttingen in Germany. According to their standard workflow cDNA libraries were prepared from protein coding mRNA that were purified through PolyA selection using the TruSeq RNA Library Prep Kit v2 Illumina. Libraries were validated using a Fragment Analyzer system Agilent Santa Clara USA before sequencing.,R1286,SAMEA10486010,"Fraunhofer Attract Eco'n'OMICs, Fraunhofer Institute for Molecular Biology and Applied Ecology, Schmallenberg, Germany",ENA first public:2022 01 01|ENA last update:2022 01 01|External Id:SAMEA10486010|INSDC center alias:Fraunhofer Attract Eco'n'OMICs Fraunhofer Institute for Molecular Biology and Applied Ecology Schmallenberg Germany|INSDC center name:Fraunhofer Attract Eco'n'OMICs Fraunhofer Institute for Molecular Biology and Applied Ecology Schmallenberg Germany|INSDC first public:2022 01 01T00:13:13Z|INSDC last update:2022 01 01T00:13:13Z|INSDC status:public|Submitter Id:E MTAB 11092:R1286|age:51|broker name:ArrayExpress|common name:zebrafish|developmental stage:hatching long pec|genotype:wild type genotype|organism part:whole organism|replicate:T13|sample name:E MTAB 11092:R1286|sex:mixed|stimulus:water|strain:AB,,,,,,,,,Illumina HiSeq 4000 sequencing; Transcriptomic profiling of clobetasol propionate induced immunosuppression in challenged zebrafish embryos,E MTAB 11092:R1286 s,R1286 s,Transcriptomic profiling of clobetasol propionate induced immunosuppression in challenged zebrafish embryos,For each sample 10 zebrafish larvae were randomly picked from the glass well and carefully transferred to a 1.5 ml low binding Eppendorf tube. Once individuals were collected for all samples the supernatant was removed and the tubes were snap frozen in liquid nitrogen. For detailed information about embryonic incubation conditions and husbandry of adult broodstock please refer to the Growth protocol. For detailed information about exposure duration and conditions please refer to the Treatment protocol. Adult animal husbandry: Wild type zebrafish Danio rerio Strain AB broodstocks were maintained under flow through conditions in 150 L tanks at 26 +/ 2C on a 12:12 h light/dark cycle. They were fed daily with TetraMin Tetra Werke Melle Germany main feed ad libitum and nauplii of Artemia salina. The day before test start glass spawning trays with artificial substrate green glass beads stringed on stainless steel wire were placed at the bottom of each tank. post mating and spawning in the morning hours eggs were rinsed with clear Cu reduced water and placed into glass dishes for pre picking. Fish embryo incubation: For each sample 20 fertilized eggs in the early blastula stage were placed in glass petri dishes diameter 6 cm filled with 8 mL of the testing solution or control medium. Embryos were incubated at 27 +/ 1C on a 14:10 h light/dark cycle. At 24 hpf eggs were inspected while half of the test solution was renewed. Clobetasol propionate CAS: 25122 46 7 was purchased from Sigma Aldrich Taufkirchen Germany. For the test solution preparation first a 5 mM stock solution was prepared in Acetone 99.9% MS grade. This stock was diluted to 250 M in acetone before 500 L were added to a 500 mL DURAN glass bottle. To the bottle for the control solution only 500 L of pure Acetone was added. post complete evaporation of the acetone the bottle was filled up with 500 mL copper reduced tap water. Complete dissolution of precipitated clobetasol propionate was achieved by sonication for 30 minutes Sonorex RK 100H Bandelin Germany. The test solutions were freshly prepared one day prior the start of the experiment and were constantly aerated at room temperature. The glass wells for the incubation of the embryos were pre saturated with the respective test solution overnight and renewed at the beginning of the experiment. Per sample about 40 50 fertilized fish eggs were pre picked in glass petri dishes 10 cm diameter filled with 30 ml of the testing solution. Using an optical binokular microscope 20 embryos of the same early blastula stage were transferred from the pre picking plate into the test glass wells filled with 8 ml of the test solution. Embryos were then incubated as described in the growth protocol. Standard flagellin from Bacillus subtilis 10% purity Pam3CSK4 98 % purity and PolyI:C HMW high molecular weight were purchased from InvivoGen SAS Toulouse France. Stock solutions 1 mg/mL for polyI:C and Pam3CSK4; 0.1 mg/mL for flagellin were prepared in provided endotoxin free water aliquoted and stored according to the manufacturers instructions until further use. At approximately 46 hpf the embryos were manually dechorinated and then further incubated. A mixture of the different PAMPs was prepared by mixing 10 L of each component resulting in final concentrations of 0.33 mg/mL for polyI:C and Pam3CSK4 and 0.033 mg/mL for flagellin respectively. The device for microinjection was set up using FemtoTip II injection needles with a complementary universal capillary holder Eppendorf Hamburg Germany connected to an IM 300 microinjector Narishige London United Kingdom. Volume calibration was performed according to Sive et al. 2010 using a micrometer slide with 1/100 mm scale Bresser Rhede Germany. Briefly the needles were backfilled with 10 L PAMP mix or ultra pure water. Injections for volume calibration were made into a drop of immersion oil purred on the micrometer slide the output pressure was constantly kept at 10 psi while the time of injection was adjusted until an injected drop had a diameter of approximately 250 m. Assuming that the drop forms a perfect sphere this corresponds to a volume of about 8 nL. The procedure described below was performed for each of the five conditions in turn treating the three replicates simultaneously. In order to anaesthetize the embryos tricaine Sigma Aldrich Taufkirchen Germany stock solution 4 mg/mL in 21 mM Tris pH 9 was added to the petri dishes to a final concentration of 200 g/mL. The immobilized embryos were transferred onto a 1 % agarose plate which was divided into segments one for each replicate. Excess liquid was removed so that the embryos were kept in place by surface tension. Desiccation was prevented by adding liquid if needed. To ensure that the injections could be performed as quickly as possible the embryos were arranged in a row in a way that their dorsal sides pointed to the same direction. The puncture was made on the dorsal side of the yolk sac and the needle was advanced through the yolk until it reached the yolk sac circulation valley on the ventral side of the embryo where the injection was made directly into the bloodstream. post all replicates of a certain condition were processed the embryos were rinsed back into the corresponding dish containing renewed test solution. Total RNA was extracted using the NucleoSpin RNA/Protein kit from Macherey Nagel 740933.250 The pooled and snap frozen larvae were suspended in RP1 buffer and were transferred to a screw cap tubes filled with 0.3 g of Lysing Matrix D MP Biomedicals 6913050 ceramic beads. Tissue homogenization was performed at 5 m/s for 45 s with FastPrep 24 MP Biomedicals 6004500 at room temperature. From here on RNA was extracted according to the manufacturers protocol. RNA concentration >100 ng/l was measured using a Nanodrop 2000 spectrophotometer Thermo Scientific. The samples overall RNA quality and corresponding RIN value was fluorometrically determined with a 2100 Bioanalyzer Instrument system Agilent G2939BA using the Agilent RNA 6000 Nano Kit Agilent 5067 1511 according to the manufacturers protocol. Only samples with a RIN > 8 were used for downstream analysis. Samples were stored at 80C until they were send to the sequencing facility on dry ice. Sequencing libraries were prepared for each sample from 100 ng/l total RNA at the sequencing facility NGS Services for Integrative Genomics at the University of Gttingen in Germany. According to their standard workflow cDNA libraries were prepared from protein coding mRNA that were purified through PolyA selection using the TruSeq RNA Library Prep Kit v2 Illumina. Libraries were validated using a Fragment Analyzer system Agilent Santa Clara USA before sequencing.,Experimental Factor: compound:Clobetasol propionate|Experimental Factor: dose:250|Experimental Factor: stimulus:water,RNA-Seq,TRANSCRIPTOMIC,PolyA,SINGLE,ILLUMINA,Illumina HiSeq 4000,,ERP132743,Illumina HiSeq 4000 sequencing; Transcriptomic profiling of clobetasol propionate induced immunosuppression in challenged zebrafish embryos,ENA FIRST PUBLIC:2022 02 25|ENA LAST UPDATE:2022 02 25|loader:fastq load.py,p946sR1286_sr.fastq.gz,fastq,2816827872.0,55797255.0,E MTAB 11092:R1286,0:50.48,A:736348159;C:671023504;G:643914486;T:761242852;N:4298871,50,,,,736348159,671023504,643914486,761242852,4298871,ERX6749455,ERS8138761,ERA6860152,Fraunhofer Attract Eco,Fraunhofer Attract Eco,1,0.94033,,0.11711,,0.68477,,0.46969,,50,,B,,usable mapping rate,illumina,hiseq_era,unknown,poly_a,trueseq,bulk,unknown,unknown,,Germany,2022-01-01,Hatching,Embryo,Whole Organism,All anatomical structures
10316,ERR7179897,ERX6749454,ERS8138760,ERP132743,PRJEB48372,Transcriptomic profiling of clobetasol propionate induced immunosuppression in challenged zebrafish embryos,E-MTAB-11092,Transcriptome Analysis,The aim of this experiment was to develop an approach for the assessment of immunotoxicity in zebrafish embryos Danio rerio using transcriptomic profiling. Clobetasol propionate a highly potent synthetic corticosteroid which is known to have immunosuppressive effects on vertebrates including zebrafish was used for continuous exposure from fertilization until extraction. As immunotoxicity can only be comprehensively assessed when the effect is observed during an acute infection the embryos were injected with a mixture of different pathogen associated molecular patterns PAMPs consisting of Pam3CSK4 synthetic lipopeptide polyI:C synthetic viral dsRNA and flagellin isolated from Bacillus subtilis. Functional gene expression analysis from samples obtained by different kinds and combinations of treatments in relation to appropriate controls is used to identify pathways that are upregulated by artificial infection and suppressed by treatment with clobetasol propionate. In a modified version of the zebrafish embryo toxicity test OECD 236 20 fertilized eggs were exposed to a sub lethal concentration 250 nM of clobetasol propionate for xxx hours under semi static conditions. The test comprised of three treatments with clobetasol propionate and two negative control NC groups and was performed in triplicates. At 46 hpf the eggs were manually dechorinated. At 48 hpf the embryos were immobilized by anaesthetization using 200 mg/mL tricaine. Using microinjection 8 nL of the mixed PAMPs 0.33 µg/mL Pam3CSK4 0.33 µg/mL polyI:C and 0.033 µg/mL flagellin were injected directly into the embryos bloodstream. One of each exposure groups was injected with PAMPs while another one was injected with ultrapure water to control for effects introduced by the injection process. One control group remained completely untreated. post injection the embryos were further incubated for 3 hours before 10 embryos were randomly picked for each sample and pooled for RNA and protein extraction with NucleoSpin RNA/Protein kit Macherey Nagel. RNA quality was assessed with a 2100 Bioanalyzer system Agilent before coding RNA was purified PolyA selection with TruSeq RNA Library Prep Kit v2 and sequenced on an Illumina HiSeq 4000 System Illumina in 50 bp single read mode. Adapter sequences were removed with trimmomatic and sequences were aligned to the D.rerio reference genome GRCz11 with STAR. Counting of feature mapped reads was performed through featureCounts. Library gene count tables were then merged to a single count matrix as input for differential gene expression analysis with DESeq2.,ENA FIRST PUBLIC:2022 02 25|ENA LAST UPDATE:2022 02 25,,Protocols: For each sample 10 zebrafish larvae were randomly picked from the glass well and carefully transferred to a 1.5 ml low binding Eppendorf tube. Once individuals were collected for all samples the supernatant was removed and the tubes were snap frozen in liquid nitrogen. For detailed information about embryonic incubation conditions and husbandry of adult broodstock please refer to the “Growth protocol”. For detailed information about exposure duration and conditions please refer to the “Treatment protocol”. Adult animal husbandry: Wild type zebrafish Danio rerio Strain AB broodstocks were maintained under flow through conditions in 150 L tanks at 26 +/ 2°C on a 12:12 h light/dark cycle. They were fed daily with TetraMin® Tetra Werke Melle Germany main feed ad libitum and nauplii of Artemia salina. The day before test start glass spawning trays with artificial substrate green glass beads stringed on stainless steel wire were placed at the bottom of each tank. post mating and spawning in the morning hours eggs were rinsed with clear Cu reduced water and placed into glass dishes for pre picking. Fish embryo incubation: For each sample 20 fertilized eggs in the early blastula stage were placed in glass petri dishes diameter 6 cm filled with 8 mL of the testing solution or control medium. Embryos were incubated at 27 +/ 1°C on a 14:10 h light/dark cycle. At 24 hpf eggs were inspected while half of the test solution was renewed. Clobetasol propionate CAS: 25122 46 7 was purchased from Sigma Aldrich Taufkirchen Germany. For the test solution preparation first a 5 mM stock solution was prepared in Acetone 99.9% MS grade. This stock was diluted to 250 µM in acetone before 500 µL were added to a 500 mL DURAN glass bottle. To the bottle for the control solution only 500 µL of pure Acetone was added. post complete evaporation of the acetone the bottle was filled up with 500 mL copper reduced tap water. Complete dissolution of precipitated clobetasol propionate was achieved by sonication for 30 minutes Sonorex RK 100H Bandelin Germany. The test solutions were freshly prepared one day prior the start of the experiment and were constantly aerated at room temperature. The glass wells for the incubation of the embryos were pre saturated with the respective test solution overnight and renewed at the beginning of the experiment. Per sample about 40 50 fertilized fish eggs were pre picked in glass petri dishes 10 cm diameter filled with 30 ml of the testing solution. Using an optical binokular microscope 20 embryos of the same early blastula stage were transferred from the pre picking plate into the test glass wells filled with 8 ml of the test solution. Embryos were then incubated as described in the “growth protocol”. Standard flagellin from Bacillus subtilis ≈ 10% purity Pam3CSK4 ≥ 98 % purity and PolyI:C HMW high molecular weight were purchased from InvivoGen SAS Toulouse France. Stock solutions 1 mg/mL for polyI:C and Pam3CSK4; 0.1 mg/mL for flagellin were prepared in provided endotoxin free water aliquoted and stored according to the manufacturer's instructions until further use. At approximately 46 hpf the embryos were manually dechorinated and then further incubated. A mixture of the different PAMPs was prepared by mixing 10 µL of each component resulting in final concentrations of 0.33 mg/mL for polyI:C and Pam3CSK4 and 0.033 mg/mL for flagellin respectively. The device for microinjection was set up using FemtoTip II injection needles with a complementary universal capillary holder Eppendorf Hamburg Germany connected to an IM 300 microinjector Narishige London United Kingdom. Volume calibration was performed according to Sive et al. 2010 using a micrometer slide with 1/100 mm scale Bresser Rhede Germany. Briefly the needles were backfilled with 10 µL PAMP mix or ultra pure water. Injections for volume calibration were made into a drop of immersion oil purred on the micrometer slide the output pressure was constantly kept at 10 psi while the time of injection was adjusted until an injected drop had a diameter of approximately 250 µm. Assuming that the drop forms a perfect sphere this corresponds to a volume of about 8 nL. The procedure described below was performed for each of the five conditions in turn treating the three replicates simultaneously. In order to anaesthetize the embryos tricaine Sigma Aldrich Taufkirchen Germany stock solution 4 mg/mL in 21 mM Tris pH 9 was added to the petri dishes to a final concentration of 200 µg/mL. The immobilized embryos were transferred onto a 1 % agarose plate which was divided into segments one for each replicate. Excess liquid was removed so that the embryos were kept in place by surface tension. Desiccation was prevented by adding liquid if needed. To ensure that the injections could be performed as quickly as possible the embryos were arranged in a row in a way that their dorsal sides pointed to the same direction. The puncture was made on the dorsal side of the yolk sac and the needle was advanced through the yolk until it reached the yolk sac circulation valley on the ventral side of the embryo where the injection was made directly into the bloodstream. post all replicates of a certain condition were processed the embryos were rinsed back into the corresponding dish containing renewed test solution. Total RNA was extracted using the NucleoSpin RNA/Protein kit from Macherey Nagel 740933.250 The pooled and snap frozen larvae were suspended in RP1 buffer and were transferred to a screw cap tubes filled with 0.3 g of Lysing Matrix D MP Biomedicals 6913050 ceramic beads. Tissue homogenization was performed at 5 m/s for 45 s with FastPrep 24© MP Biomedicals 6004500 at room temperature. From here on RNA was extracted according to the manufacturer's protocol. RNA concentration >100 ng/µl was measured using a Nanodrop 2000 spectrophotometer Thermo Scientific. The sample's overall RNA quality and corresponding RIN value was fluorometrically determined with a 2100 Bioanalyzer© Instrument system Agilent G2939BA using the Agilent RNA 6000 Nano Kit© Agilent 5067 1511 according to the manufacturer's protocol. Only samples with a RIN > 8 were used for downstream analysis. Samples were stored at 80°C until they were send to the sequencing facility on dry ice. Sequencing libraries were prepared for each sample from 100 ng/µl total RNA at the sequencing facility “NGS Services for Integrative Genomics” at the University of Göttingen in Germany. According to their standard workflow cDNA libraries were prepared from protein coding mRNA that were purified through PolyA selection using the TruSeq RNA Library Prep Kit v2 Illumina. Libraries were validated using a Fragment Analyzer system Agilent Santa Clara USA before sequencing.,R1277,SAMEA10486008,"Fraunhofer Attract Eco'n'OMICs, Fraunhofer Institute for Molecular Biology and Applied Ecology, Schmallenberg, Germany",ENA first public:2022 01 01|ENA last update:2022 01 01|External Id:SAMEA10486008|INSDC center alias:Fraunhofer Attract Eco'n'OMICs Fraunhofer Institute for Molecular Biology and Applied Ecology Schmallenberg Germany|INSDC center name:Fraunhofer Attract Eco'n'OMICs Fraunhofer Institute for Molecular Biology and Applied Ecology Schmallenberg Germany|INSDC first public:2022 01 01T00:13:13Z|INSDC last update:2022 01 01T00:13:13Z|INSDC status:public|Submitter Id:E MTAB 11092:R1277|age:51|broker name:ArrayExpress|common name:zebrafish|developmental stage:hatching long pec|genotype:wild type genotype|organism part:whole organism|replicate:T13|sample name:E MTAB 11092:R1277|sex:mixed|stimulus:PAMP|strain:AB,,,,,,,,,Illumina HiSeq 4000 sequencing; Transcriptomic profiling of clobetasol propionate induced immunosuppression in challenged zebrafish embryos,E MTAB 11092:R1277 s,R1277 s,Transcriptomic profiling of clobetasol propionate induced immunosuppression in challenged zebrafish embryos,For each sample 10 zebrafish larvae were randomly picked from the glass well and carefully transferred to a 1.5 ml low binding Eppendorf tube. Once individuals were collected for all samples the supernatant was removed and the tubes were snap frozen in liquid nitrogen. For detailed information about embryonic incubation conditions and husbandry of adult broodstock please refer to the Growth protocol. For detailed information about exposure duration and conditions please refer to the Treatment protocol. Adult animal husbandry: Wild type zebrafish Danio rerio Strain AB broodstocks were maintained under flow through conditions in 150 L tanks at 26 +/ 2C on a 12:12 h light/dark cycle. They were fed daily with TetraMin Tetra Werke Melle Germany main feed ad libitum and nauplii of Artemia salina. The day before test start glass spawning trays with artificial substrate green glass beads stringed on stainless steel wire were placed at the bottom of each tank. post mating and spawning in the morning hours eggs were rinsed with clear Cu reduced water and placed into glass dishes for pre picking. Fish embryo incubation: For each sample 20 fertilized eggs in the early blastula stage were placed in glass petri dishes diameter 6 cm filled with 8 mL of the testing solution or control medium. Embryos were incubated at 27 +/ 1C on a 14:10 h light/dark cycle. At 24 hpf eggs were inspected while half of the test solution was renewed. Clobetasol propionate CAS: 25122 46 7 was purchased from Sigma Aldrich Taufkirchen Germany. For the test solution preparation first a 5 mM stock solution was prepared in Acetone 99.9% MS grade. This stock was diluted to 250 M in acetone before 500 L were added to a 500 mL DURAN glass bottle. To the bottle for the control solution only 500 L of pure Acetone was added. post complete evaporation of the acetone the bottle was filled up with 500 mL copper reduced tap water. Complete dissolution of precipitated clobetasol propionate was achieved by sonication for 30 minutes Sonorex RK 100H Bandelin Germany. The test solutions were freshly prepared one day prior the start of the experiment and were constantly aerated at room temperature. The glass wells for the incubation of the embryos were pre saturated with the respective test solution overnight and renewed at the beginning of the experiment. Per sample about 40 50 fertilized fish eggs were pre picked in glass petri dishes 10 cm diameter filled with 30 ml of the testing solution. Using an optical binokular microscope 20 embryos of the same early blastula stage were transferred from the pre picking plate into the test glass wells filled with 8 ml of the test solution. Embryos were then incubated as described in the growth protocol. Standard flagellin from Bacillus subtilis 10% purity Pam3CSK4 98 % purity and PolyI:C HMW high molecular weight were purchased from InvivoGen SAS Toulouse France. Stock solutions 1 mg/mL for polyI:C and Pam3CSK4; 0.1 mg/mL for flagellin were prepared in provided endotoxin free water aliquoted and stored according to the manufacturers instructions until further use. At approximately 46 hpf the embryos were manually dechorinated and then further incubated. A mixture of the different PAMPs was prepared by mixing 10 L of each component resulting in final concentrations of 0.33 mg/mL for polyI:C and Pam3CSK4 and 0.033 mg/mL for flagellin respectively. The device for microinjection was set up using FemtoTip II injection needles with a complementary universal capillary holder Eppendorf Hamburg Germany connected to an IM 300 microinjector Narishige London United Kingdom. Volume calibration was performed according to Sive et al. 2010 using a micrometer slide with 1/100 mm scale Bresser Rhede Germany. Briefly the needles were backfilled with 10 L PAMP mix or ultra pure water. Injections for volume calibration were made into a drop of immersion oil purred on the micrometer slide the output pressure was constantly kept at 10 psi while the time of injection was adjusted until an injected drop had a diameter of approximately 250 m. Assuming that the drop forms a perfect sphere this corresponds to a volume of about 8 nL. The procedure described below was performed for each of the five conditions in turn treating the three replicates simultaneously. In order to anaesthetize the embryos tricaine Sigma Aldrich Taufkirchen Germany stock solution 4 mg/mL in 21 mM Tris pH 9 was added to the petri dishes to a final concentration of 200 g/mL. The immobilized embryos were transferred onto a 1 % agarose plate which was divided into segments one for each replicate. Excess liquid was removed so that the embryos were kept in place by surface tension. Desiccation was prevented by adding liquid if needed. To ensure that the injections could be performed as quickly as possible the embryos were arranged in a row in a way that their dorsal sides pointed to the same direction. The puncture was made on the dorsal side of the yolk sac and the needle was advanced through the yolk until it reached the yolk sac circulation valley on the ventral side of the embryo where the injection was made directly into the bloodstream. post all replicates of a certain condition were processed the embryos were rinsed back into the corresponding dish containing renewed test solution. Total RNA was extracted using the NucleoSpin RNA/Protein kit from Macherey Nagel 740933.250 The pooled and snap frozen larvae were suspended in RP1 buffer and were transferred to a screw cap tubes filled with 0.3 g of Lysing Matrix D MP Biomedicals 6913050 ceramic beads. Tissue homogenization was performed at 5 m/s for 45 s with FastPrep 24 MP Biomedicals 6004500 at room temperature. From here on RNA was extracted according to the manufacturers protocol. RNA concentration >100 ng/l was measured using a Nanodrop 2000 spectrophotometer Thermo Scientific. The samples overall RNA quality and corresponding RIN value was fluorometrically determined with a 2100 Bioanalyzer Instrument system Agilent G2939BA using the Agilent RNA 6000 Nano Kit Agilent 5067 1511 according to the manufacturers protocol. Only samples with a RIN > 8 were used for downstream analysis. Samples were stored at 80C until they were send to the sequencing facility on dry ice. Sequencing libraries were prepared for each sample from 100 ng/l total RNA at the sequencing facility NGS Services for Integrative Genomics at the University of Gttingen in Germany. According to their standard workflow cDNA libraries were prepared from protein coding mRNA that were purified through PolyA selection using the TruSeq RNA Library Prep Kit v2 Illumina. Libraries were validated using a Fragment Analyzer system Agilent Santa Clara USA before sequencing.,Experimental Factor: compound:n1|Experimental Factor: stimulus:PAMP,RNA-Seq,TRANSCRIPTOMIC,PolyA,SINGLE,ILLUMINA,Illumina HiSeq 4000,,ERP132743,Illumina HiSeq 4000 sequencing; Transcriptomic profiling of clobetasol propionate induced immunosuppression in challenged zebrafish embryos,ENA FIRST PUBLIC:2022 02 25|ENA LAST UPDATE:2022 02 25,p946sR1277_sr.fastq.gz,fastq,1268705881.0,25118834.0,E MTAB 11092:R1277,0:50.51 1:0,A:329102604;C:306045954;G:293488785;T:339279603;N:788935,50,0,,,329102604,306045954,293488785,339279603,788935,ERX6749454,ERS8138760,ERA6860152,Fraunhofer Attract Eco,Fraunhofer Attract Eco,1,0.94045,,0.11288,,0.69025,,0.47095,,48,,B,,usable mapping rate,illumina,hiseq_era,unknown,poly_a,trueseq,bulk,unknown,unknown,,Germany,2022-01-01,Hatching,Embryo,Whole Organism,All anatomical structures
10317,ERR7179896,ERX6749453,ERS8138759,ERP132743,PRJEB48372,Transcriptomic profiling of clobetasol propionate induced immunosuppression in challenged zebrafish embryos,E-MTAB-11092,Transcriptome Analysis,The aim of this experiment was to develop an approach for the assessment of immunotoxicity in zebrafish embryos Danio rerio using transcriptomic profiling. Clobetasol propionate a highly potent synthetic corticosteroid which is known to have immunosuppressive effects on vertebrates including zebrafish was used for continuous exposure from fertilization until extraction. As immunotoxicity can only be comprehensively assessed when the effect is observed during an acute infection the embryos were injected with a mixture of different pathogen associated molecular patterns PAMPs consisting of Pam3CSK4 synthetic lipopeptide polyI:C synthetic viral dsRNA and flagellin isolated from Bacillus subtilis. Functional gene expression analysis from samples obtained by different kinds and combinations of treatments in relation to appropriate controls is used to identify pathways that are upregulated by artificial infection and suppressed by treatment with clobetasol propionate. In a modified version of the zebrafish embryo toxicity test OECD 236 20 fertilized eggs were exposed to a sub lethal concentration 250 nM of clobetasol propionate for xxx hours under semi static conditions. The test comprised of three treatments with clobetasol propionate and two negative control NC groups and was performed in triplicates. At 46 hpf the eggs were manually dechorinated. At 48 hpf the embryos were immobilized by anaesthetization using 200 mg/mL tricaine. Using microinjection 8 nL of the mixed PAMPs 0.33 µg/mL Pam3CSK4 0.33 µg/mL polyI:C and 0.033 µg/mL flagellin were injected directly into the embryos bloodstream. One of each exposure groups was injected with PAMPs while another one was injected with ultrapure water to control for effects introduced by the injection process. One control group remained completely untreated. post injection the embryos were further incubated for 3 hours before 10 embryos were randomly picked for each sample and pooled for RNA and protein extraction with NucleoSpin RNA/Protein kit Macherey Nagel. RNA quality was assessed with a 2100 Bioanalyzer system Agilent before coding RNA was purified PolyA selection with TruSeq RNA Library Prep Kit v2 and sequenced on an Illumina HiSeq 4000 System Illumina in 50 bp single read mode. Adapter sequences were removed with trimmomatic and sequences were aligned to the D.rerio reference genome GRCz11 with STAR. Counting of feature mapped reads was performed through featureCounts. Library gene count tables were then merged to a single count matrix as input for differential gene expression analysis with DESeq2.,ENA FIRST PUBLIC:2022 02 25|ENA LAST UPDATE:2022 02 25,,Protocols: For each sample 10 zebrafish larvae were randomly picked from the glass well and carefully transferred to a 1.5 ml low binding Eppendorf tube. Once individuals were collected for all samples the supernatant was removed and the tubes were snap frozen in liquid nitrogen. For detailed information about embryonic incubation conditions and husbandry of adult broodstock please refer to the “Growth protocol”. For detailed information about exposure duration and conditions please refer to the “Treatment protocol”. Adult animal husbandry: Wild type zebrafish Danio rerio Strain AB broodstocks were maintained under flow through conditions in 150 L tanks at 26 +/ 2°C on a 12:12 h light/dark cycle. They were fed daily with TetraMin® Tetra Werke Melle Germany main feed ad libitum and nauplii of Artemia salina. The day before test start glass spawning trays with artificial substrate green glass beads stringed on stainless steel wire were placed at the bottom of each tank. post mating and spawning in the morning hours eggs were rinsed with clear Cu reduced water and placed into glass dishes for pre picking. Fish embryo incubation: For each sample 20 fertilized eggs in the early blastula stage were placed in glass petri dishes diameter 6 cm filled with 8 mL of the testing solution or control medium. Embryos were incubated at 27 +/ 1°C on a 14:10 h light/dark cycle. At 24 hpf eggs were inspected while half of the test solution was renewed. Clobetasol propionate CAS: 25122 46 7 was purchased from Sigma Aldrich Taufkirchen Germany. For the test solution preparation first a 5 mM stock solution was prepared in Acetone 99.9% MS grade. This stock was diluted to 250 µM in acetone before 500 µL were added to a 500 mL DURAN glass bottle. To the bottle for the control solution only 500 µL of pure Acetone was added. post complete evaporation of the acetone the bottle was filled up with 500 mL copper reduced tap water. Complete dissolution of precipitated clobetasol propionate was achieved by sonication for 30 minutes Sonorex RK 100H Bandelin Germany. The test solutions were freshly prepared one day prior the start of the experiment and were constantly aerated at room temperature. The glass wells for the incubation of the embryos were pre saturated with the respective test solution overnight and renewed at the beginning of the experiment. Per sample about 40 50 fertilized fish eggs were pre picked in glass petri dishes 10 cm diameter filled with 30 ml of the testing solution. Using an optical binokular microscope 20 embryos of the same early blastula stage were transferred from the pre picking plate into the test glass wells filled with 8 ml of the test solution. Embryos were then incubated as described in the “growth protocol”. Standard flagellin from Bacillus subtilis ≈ 10% purity Pam3CSK4 ≥ 98 % purity and PolyI:C HMW high molecular weight were purchased from InvivoGen SAS Toulouse France. Stock solutions 1 mg/mL for polyI:C and Pam3CSK4; 0.1 mg/mL for flagellin were prepared in provided endotoxin free water aliquoted and stored according to the manufacturer's instructions until further use. At approximately 46 hpf the embryos were manually dechorinated and then further incubated. A mixture of the different PAMPs was prepared by mixing 10 µL of each component resulting in final concentrations of 0.33 mg/mL for polyI:C and Pam3CSK4 and 0.033 mg/mL for flagellin respectively. The device for microinjection was set up using FemtoTip II injection needles with a complementary universal capillary holder Eppendorf Hamburg Germany connected to an IM 300 microinjector Narishige London United Kingdom. Volume calibration was performed according to Sive et al. 2010 using a micrometer slide with 1/100 mm scale Bresser Rhede Germany. Briefly the needles were backfilled with 10 µL PAMP mix or ultra pure water. Injections for volume calibration were made into a drop of immersion oil purred on the micrometer slide the output pressure was constantly kept at 10 psi while the time of injection was adjusted until an injected drop had a diameter of approximately 250 µm. Assuming that the drop forms a perfect sphere this corresponds to a volume of about 8 nL. The procedure described below was performed for each of the five conditions in turn treating the three replicates simultaneously. In order to anaesthetize the embryos tricaine Sigma Aldrich Taufkirchen Germany stock solution 4 mg/mL in 21 mM Tris pH 9 was added to the petri dishes to a final concentration of 200 µg/mL. The immobilized embryos were transferred onto a 1 % agarose plate which was divided into segments one for each replicate. Excess liquid was removed so that the embryos were kept in place by surface tension. Desiccation was prevented by adding liquid if needed. To ensure that the injections could be performed as quickly as possible the embryos were arranged in a row in a way that their dorsal sides pointed to the same direction. The puncture was made on the dorsal side of the yolk sac and the needle was advanced through the yolk until it reached the yolk sac circulation valley on the ventral side of the embryo where the injection was made directly into the bloodstream. post all replicates of a certain condition were processed the embryos were rinsed back into the corresponding dish containing renewed test solution. Total RNA was extracted using the NucleoSpin RNA/Protein kit from Macherey Nagel 740933.250 The pooled and snap frozen larvae were suspended in RP1 buffer and were transferred to a screw cap tubes filled with 0.3 g of Lysing Matrix D MP Biomedicals 6913050 ceramic beads. Tissue homogenization was performed at 5 m/s for 45 s with FastPrep 24© MP Biomedicals 6004500 at room temperature. From here on RNA was extracted according to the manufacturer's protocol. RNA concentration >100 ng/µl was measured using a Nanodrop 2000 spectrophotometer Thermo Scientific. The sample's overall RNA quality and corresponding RIN value was fluorometrically determined with a 2100 Bioanalyzer© Instrument system Agilent G2939BA using the Agilent RNA 6000 Nano Kit© Agilent 5067 1511 according to the manufacturer's protocol. Only samples with a RIN > 8 were used for downstream analysis. Samples were stored at 80°C until they were send to the sequencing facility on dry ice. Sequencing libraries were prepared for each sample from 100 ng/µl total RNA at the sequencing facility “NGS Services for Integrative Genomics” at the University of Göttingen in Germany. According to their standard workflow cDNA libraries were prepared from protein coding mRNA that were purified through PolyA selection using the TruSeq RNA Library Prep Kit v2 Illumina. Libraries were validated using a Fragment Analyzer system Agilent Santa Clara USA before sequencing.,R1274,SAMEA10486006,"Fraunhofer Attract Eco'n'OMICs, Fraunhofer Institute for Molecular Biology and Applied Ecology, Schmallenberg, Germany",ENA first public:2022 01 01|ENA last update:2022 01 01|External Id:SAMEA10486006|INSDC center alias:Fraunhofer Attract Eco'n'OMICs Fraunhofer Institute for Molecular Biology and Applied Ecology Schmallenberg Germany|INSDC center name:Fraunhofer Attract Eco'n'OMICs Fraunhofer Institute for Molecular Biology and Applied Ecology Schmallenberg Germany|INSDC first public:2022 01 01T00:13:13Z|INSDC last update:2022 01 01T00:13:13Z|INSDC status:public|Submitter Id:E MTAB 11092:R1274|age:51|broker name:ArrayExpress|common name:zebrafish|developmental stage:hatching long pec|genotype:wild type genotype|organism part:whole organism|replicate:T13|sample name:E MTAB 11092:R1274|sex:mixed|stimulus:water|strain:AB,,,,,,,,,Illumina HiSeq 4000 sequencing; Transcriptomic profiling of clobetasol propionate induced immunosuppression in challenged zebrafish embryos,E MTAB 11092:R1274 s,R1274 s,Transcriptomic profiling of clobetasol propionate induced immunosuppression in challenged zebrafish embryos,For each sample 10 zebrafish larvae were randomly picked from the glass well and carefully transferred to a 1.5 ml low binding Eppendorf tube. Once individuals were collected for all samples the supernatant was removed and the tubes were snap frozen in liquid nitrogen. For detailed information about embryonic incubation conditions and husbandry of adult broodstock please refer to the Growth protocol. For detailed information about exposure duration and conditions please refer to the Treatment protocol. Adult animal husbandry: Wild type zebrafish Danio rerio Strain AB broodstocks were maintained under flow through conditions in 150 L tanks at 26 +/ 2C on a 12:12 h light/dark cycle. They were fed daily with TetraMin Tetra Werke Melle Germany main feed ad libitum and nauplii of Artemia salina. The day before test start glass spawning trays with artificial substrate green glass beads stringed on stainless steel wire were placed at the bottom of each tank. post mating and spawning in the morning hours eggs were rinsed with clear Cu reduced water and placed into glass dishes for pre picking. Fish embryo incubation: For each sample 20 fertilized eggs in the early blastula stage were placed in glass petri dishes diameter 6 cm filled with 8 mL of the testing solution or control medium. Embryos were incubated at 27 +/ 1C on a 14:10 h light/dark cycle. At 24 hpf eggs were inspected while half of the test solution was renewed. Clobetasol propionate CAS: 25122 46 7 was purchased from Sigma Aldrich Taufkirchen Germany. For the test solution preparation first a 5 mM stock solution was prepared in Acetone 99.9% MS grade. This stock was diluted to 250 M in acetone before 500 L were added to a 500 mL DURAN glass bottle. To the bottle for the control solution only 500 L of pure Acetone was added. post complete evaporation of the acetone the bottle was filled up with 500 mL copper reduced tap water. Complete dissolution of precipitated clobetasol propionate was achieved by sonication for 30 minutes Sonorex RK 100H Bandelin Germany. The test solutions were freshly prepared one day prior the start of the experiment and were constantly aerated at room temperature. The glass wells for the incubation of the embryos were pre saturated with the respective test solution overnight and renewed at the beginning of the experiment. Per sample about 40 50 fertilized fish eggs were pre picked in glass petri dishes 10 cm diameter filled with 30 ml of the testing solution. Using an optical binokular microscope 20 embryos of the same early blastula stage were transferred from the pre picking plate into the test glass wells filled with 8 ml of the test solution. Embryos were then incubated as described in the growth protocol. Standard flagellin from Bacillus subtilis 10% purity Pam3CSK4 98 % purity and PolyI:C HMW high molecular weight were purchased from InvivoGen SAS Toulouse France. Stock solutions 1 mg/mL for polyI:C and Pam3CSK4; 0.1 mg/mL for flagellin were prepared in provided endotoxin free water aliquoted and stored according to the manufacturers instructions until further use. At approximately 46 hpf the embryos were manually dechorinated and then further incubated. A mixture of the different PAMPs was prepared by mixing 10 L of each component resulting in final concentrations of 0.33 mg/mL for polyI:C and Pam3CSK4 and 0.033 mg/mL for flagellin respectively. The device for microinjection was set up using FemtoTip II injection needles with a complementary universal capillary holder Eppendorf Hamburg Germany connected to an IM 300 microinjector Narishige London United Kingdom. Volume calibration was performed according to Sive et al. 2010 using a micrometer slide with 1/100 mm scale Bresser Rhede Germany. Briefly the needles were backfilled with 10 L PAMP mix or ultra pure water. Injections for volume calibration were made into a drop of immersion oil purred on the micrometer slide the output pressure was constantly kept at 10 psi while the time of injection was adjusted until an injected drop had a diameter of approximately 250 m. Assuming that the drop forms a perfect sphere this corresponds to a volume of about 8 nL. The procedure described below was performed for each of the five conditions in turn treating the three replicates simultaneously. In order to anaesthetize the embryos tricaine Sigma Aldrich Taufkirchen Germany stock solution 4 mg/mL in 21 mM Tris pH 9 was added to the petri dishes to a final concentration of 200 g/mL. The immobilized embryos were transferred onto a 1 % agarose plate which was divided into segments one for each replicate. Excess liquid was removed so that the embryos were kept in place by surface tension. Desiccation was prevented by adding liquid if needed. To ensure that the injections could be performed as quickly as possible the embryos were arranged in a row in a way that their dorsal sides pointed to the same direction. The puncture was made on the dorsal side of the yolk sac and the needle was advanced through the yolk until it reached the yolk sac circulation valley on the ventral side of the embryo where the injection was made directly into the bloodstream. post all replicates of a certain condition were processed the embryos were rinsed back into the corresponding dish containing renewed test solution. Total RNA was extracted using the NucleoSpin RNA/Protein kit from Macherey Nagel 740933.250 The pooled and snap frozen larvae were suspended in RP1 buffer and were transferred to a screw cap tubes filled with 0.3 g of Lysing Matrix D MP Biomedicals 6913050 ceramic beads. Tissue homogenization was performed at 5 m/s for 45 s with FastPrep 24 MP Biomedicals 6004500 at room temperature. From here on RNA was extracted according to the manufacturers protocol. RNA concentration >100 ng/l was measured using a Nanodrop 2000 spectrophotometer Thermo Scientific. The samples overall RNA quality and corresponding RIN value was fluorometrically determined with a 2100 Bioanalyzer Instrument system Agilent G2939BA using the Agilent RNA 6000 Nano Kit Agilent 5067 1511 according to the manufacturers protocol. Only samples with a RIN > 8 were used for downstream analysis. Samples were stored at 80C until they were send to the sequencing facility on dry ice. Sequencing libraries were prepared for each sample from 100 ng/l total RNA at the sequencing facility NGS Services for Integrative Genomics at the University of Gttingen in Germany. According to their standard workflow cDNA libraries were prepared from protein coding mRNA that were purified through PolyA selection using the TruSeq RNA Library Prep Kit v2 Illumina. Libraries were validated using a Fragment Analyzer system Agilent Santa Clara USA before sequencing.,Experimental Factor: compound:n1|Experimental Factor: stimulus:water,RNA-Seq,TRANSCRIPTOMIC,PolyA,SINGLE,ILLUMINA,Illumina HiSeq 4000,,ERP132743,Illumina HiSeq 4000 sequencing; Transcriptomic profiling of clobetasol propionate induced immunosuppression in challenged zebrafish embryos,ENA FIRST PUBLIC:2022 02 25|ENA LAST UPDATE:2022 02 25|loader:fastq load.py,p946sR1274_sr.fastq.gz,fastq,1107133784.0,21923109.0,E MTAB 11092:R1274,0:50.50,A:289596768;C:264193186;G:253295296;T:299245790;N:802744,50,,,,289596768,264193186,253295296,299245790,802744,ERX6749453,ERS8138759,ERA6860152,Fraunhofer Attract Eco,Fraunhofer Attract Eco,1,0.93893,,0.1111,,0.68091,,0.46331,,48,,B,,usable mapping rate,illumina,hiseq_era,unknown,poly_a,trueseq,bulk,unknown,unknown,,Germany,2022-01-01,Hatching,Embryo,Whole Organism,All anatomical structures
10318,ERR7179895,ERX6749452,ERS8138758,ERP132743,PRJEB48372,Transcriptomic profiling of clobetasol propionate induced immunosuppression in challenged zebrafish embryos,E-MTAB-11092,Transcriptome Analysis,The aim of this experiment was to develop an approach for the assessment of immunotoxicity in zebrafish embryos Danio rerio using transcriptomic profiling. Clobetasol propionate a highly potent synthetic corticosteroid which is known to have immunosuppressive effects on vertebrates including zebrafish was used for continuous exposure from fertilization until extraction. As immunotoxicity can only be comprehensively assessed when the effect is observed during an acute infection the embryos were injected with a mixture of different pathogen associated molecular patterns PAMPs consisting of Pam3CSK4 synthetic lipopeptide polyI:C synthetic viral dsRNA and flagellin isolated from Bacillus subtilis. Functional gene expression analysis from samples obtained by different kinds and combinations of treatments in relation to appropriate controls is used to identify pathways that are upregulated by artificial infection and suppressed by treatment with clobetasol propionate. In a modified version of the zebrafish embryo toxicity test OECD 236 20 fertilized eggs were exposed to a sub lethal concentration 250 nM of clobetasol propionate for xxx hours under semi static conditions. The test comprised of three treatments with clobetasol propionate and two negative control NC groups and was performed in triplicates. At 46 hpf the eggs were manually dechorinated. At 48 hpf the embryos were immobilized by anaesthetization using 200 mg/mL tricaine. Using microinjection 8 nL of the mixed PAMPs 0.33 µg/mL Pam3CSK4 0.33 µg/mL polyI:C and 0.033 µg/mL flagellin were injected directly into the embryos bloodstream. One of each exposure groups was injected with PAMPs while another one was injected with ultrapure water to control for effects introduced by the injection process. One control group remained completely untreated. post injection the embryos were further incubated for 3 hours before 10 embryos were randomly picked for each sample and pooled for RNA and protein extraction with NucleoSpin RNA/Protein kit Macherey Nagel. RNA quality was assessed with a 2100 Bioanalyzer system Agilent before coding RNA was purified PolyA selection with TruSeq RNA Library Prep Kit v2 and sequenced on an Illumina HiSeq 4000 System Illumina in 50 bp single read mode. Adapter sequences were removed with trimmomatic and sequences were aligned to the D.rerio reference genome GRCz11 with STAR. Counting of feature mapped reads was performed through featureCounts. Library gene count tables were then merged to a single count matrix as input for differential gene expression analysis with DESeq2.,ENA FIRST PUBLIC:2022 02 25|ENA LAST UPDATE:2022 02 25,,Protocols: For each sample 10 zebrafish larvae were randomly picked from the glass well and carefully transferred to a 1.5 ml low binding Eppendorf tube. Once individuals were collected for all samples the supernatant was removed and the tubes were snap frozen in liquid nitrogen. For detailed information about embryonic incubation conditions and husbandry of adult broodstock please refer to the “Growth protocol”. For detailed information about exposure duration and conditions please refer to the “Treatment protocol”. Adult animal husbandry: Wild type zebrafish Danio rerio Strain AB broodstocks were maintained under flow through conditions in 150 L tanks at 26 +/ 2°C on a 12:12 h light/dark cycle. They were fed daily with TetraMin® Tetra Werke Melle Germany main feed ad libitum and nauplii of Artemia salina. The day before test start glass spawning trays with artificial substrate green glass beads stringed on stainless steel wire were placed at the bottom of each tank. post mating and spawning in the morning hours eggs were rinsed with clear Cu reduced water and placed into glass dishes for pre picking. Fish embryo incubation: For each sample 20 fertilized eggs in the early blastula stage were placed in glass petri dishes diameter 6 cm filled with 8 mL of the testing solution or control medium. Embryos were incubated at 27 +/ 1°C on a 14:10 h light/dark cycle. At 24 hpf eggs were inspected while half of the test solution was renewed. Clobetasol propionate CAS: 25122 46 7 was purchased from Sigma Aldrich Taufkirchen Germany. For the test solution preparation first a 5 mM stock solution was prepared in Acetone 99.9% MS grade. This stock was diluted to 250 µM in acetone before 500 µL were added to a 500 mL DURAN glass bottle. To the bottle for the control solution only 500 µL of pure Acetone was added. post complete evaporation of the acetone the bottle was filled up with 500 mL copper reduced tap water. Complete dissolution of precipitated clobetasol propionate was achieved by sonication for 30 minutes Sonorex RK 100H Bandelin Germany. The test solutions were freshly prepared one day prior the start of the experiment and were constantly aerated at room temperature. The glass wells for the incubation of the embryos were pre saturated with the respective test solution overnight and renewed at the beginning of the experiment. Per sample about 40 50 fertilized fish eggs were pre picked in glass petri dishes 10 cm diameter filled with 30 ml of the testing solution. Using an optical binokular microscope 20 embryos of the same early blastula stage were transferred from the pre picking plate into the test glass wells filled with 8 ml of the test solution. Embryos were then incubated as described in the “growth protocol”. Standard flagellin from Bacillus subtilis ≈ 10% purity Pam3CSK4 ≥ 98 % purity and PolyI:C HMW high molecular weight were purchased from InvivoGen SAS Toulouse France. Stock solutions 1 mg/mL for polyI:C and Pam3CSK4; 0.1 mg/mL for flagellin were prepared in provided endotoxin free water aliquoted and stored according to the manufacturer's instructions until further use. At approximately 46 hpf the embryos were manually dechorinated and then further incubated. A mixture of the different PAMPs was prepared by mixing 10 µL of each component resulting in final concentrations of 0.33 mg/mL for polyI:C and Pam3CSK4 and 0.033 mg/mL for flagellin respectively. The device for microinjection was set up using FemtoTip II injection needles with a complementary universal capillary holder Eppendorf Hamburg Germany connected to an IM 300 microinjector Narishige London United Kingdom. Volume calibration was performed according to Sive et al. 2010 using a micrometer slide with 1/100 mm scale Bresser Rhede Germany. Briefly the needles were backfilled with 10 µL PAMP mix or ultra pure water. Injections for volume calibration were made into a drop of immersion oil purred on the micrometer slide the output pressure was constantly kept at 10 psi while the time of injection was adjusted until an injected drop had a diameter of approximately 250 µm. Assuming that the drop forms a perfect sphere this corresponds to a volume of about 8 nL. The procedure described below was performed for each of the five conditions in turn treating the three replicates simultaneously. In order to anaesthetize the embryos tricaine Sigma Aldrich Taufkirchen Germany stock solution 4 mg/mL in 21 mM Tris pH 9 was added to the petri dishes to a final concentration of 200 µg/mL. The immobilized embryos were transferred onto a 1 % agarose plate which was divided into segments one for each replicate. Excess liquid was removed so that the embryos were kept in place by surface tension. Desiccation was prevented by adding liquid if needed. To ensure that the injections could be performed as quickly as possible the embryos were arranged in a row in a way that their dorsal sides pointed to the same direction. The puncture was made on the dorsal side of the yolk sac and the needle was advanced through the yolk until it reached the yolk sac circulation valley on the ventral side of the embryo where the injection was made directly into the bloodstream. post all replicates of a certain condition were processed the embryos were rinsed back into the corresponding dish containing renewed test solution. Total RNA was extracted using the NucleoSpin RNA/Protein kit from Macherey Nagel 740933.250 The pooled and snap frozen larvae were suspended in RP1 buffer and were transferred to a screw cap tubes filled with 0.3 g of Lysing Matrix D MP Biomedicals 6913050 ceramic beads. Tissue homogenization was performed at 5 m/s for 45 s with FastPrep 24© MP Biomedicals 6004500 at room temperature. From here on RNA was extracted according to the manufacturer's protocol. RNA concentration >100 ng/µl was measured using a Nanodrop 2000 spectrophotometer Thermo Scientific. The sample's overall RNA quality and corresponding RIN value was fluorometrically determined with a 2100 Bioanalyzer© Instrument system Agilent G2939BA using the Agilent RNA 6000 Nano Kit© Agilent 5067 1511 according to the manufacturer's protocol. Only samples with a RIN > 8 were used for downstream analysis. Samples were stored at 80°C until they were send to the sequencing facility on dry ice. Sequencing libraries were prepared for each sample from 100 ng/µl total RNA at the sequencing facility “NGS Services for Integrative Genomics” at the University of Göttingen in Germany. According to their standard workflow cDNA libraries were prepared from protein coding mRNA that were purified through PolyA selection using the TruSeq RNA Library Prep Kit v2 Illumina. Libraries were validated using a Fragment Analyzer system Agilent Santa Clara USA before sequencing.,R1271,SAMEA10486004,"Fraunhofer Attract Eco'n'OMICs, Fraunhofer Institute for Molecular Biology and Applied Ecology, Schmallenberg, Germany",ENA first public:2022 01 01|ENA last update:2022 01 01|External Id:SAMEA10486004|INSDC center alias:Fraunhofer Attract Eco'n'OMICs Fraunhofer Institute for Molecular Biology and Applied Ecology Schmallenberg Germany|INSDC center name:Fraunhofer Attract Eco'n'OMICs Fraunhofer Institute for Molecular Biology and Applied Ecology Schmallenberg Germany|INSDC first public:2022 01 01T00:13:13Z|INSDC last update:2022 01 01T00:13:13Z|INSDC status:public|Submitter Id:E MTAB 11092:R1271|age:51|broker name:ArrayExpress|common name:zebrafish|developmental stage:hatching long pec|genotype:wild type genotype|organism part:whole organism|replicate:T13|sample name:E MTAB 11092:R1271|sex:mixed|stimulus:control|strain:AB,,,,,,,,,Illumina HiSeq 4000 sequencing; Transcriptomic profiling of clobetasol propionate induced immunosuppression in challenged zebrafish embryos,E MTAB 11092:R1271 s,R1271 s,Transcriptomic profiling of clobetasol propionate induced immunosuppression in challenged zebrafish embryos,For each sample 10 zebrafish larvae were randomly picked from the glass well and carefully transferred to a 1.5 ml low binding Eppendorf tube. Once individuals were collected for all samples the supernatant was removed and the tubes were snap frozen in liquid nitrogen. For detailed information about embryonic incubation conditions and husbandry of adult broodstock please refer to the Growth protocol. For detailed information about exposure duration and conditions please refer to the Treatment protocol. Adult animal husbandry: Wild type zebrafish Danio rerio Strain AB broodstocks were maintained under flow through conditions in 150 L tanks at 26 +/ 2C on a 12:12 h light/dark cycle. They were fed daily with TetraMin Tetra Werke Melle Germany main feed ad libitum and nauplii of Artemia salina. The day before test start glass spawning trays with artificial substrate green glass beads stringed on stainless steel wire were placed at the bottom of each tank. post mating and spawning in the morning hours eggs were rinsed with clear Cu reduced water and placed into glass dishes for pre picking. Fish embryo incubation: For each sample 20 fertilized eggs in the early blastula stage were placed in glass petri dishes diameter 6 cm filled with 8 mL of the testing solution or control medium. Embryos were incubated at 27 +/ 1C on a 14:10 h light/dark cycle. At 24 hpf eggs were inspected while half of the test solution was renewed. Clobetasol propionate CAS: 25122 46 7 was purchased from Sigma Aldrich Taufkirchen Germany. For the test solution preparation first a 5 mM stock solution was prepared in Acetone 99.9% MS grade. This stock was diluted to 250 M in acetone before 500 L were added to a 500 mL DURAN glass bottle. To the bottle for the control solution only 500 L of pure Acetone was added. post complete evaporation of the acetone the bottle was filled up with 500 mL copper reduced tap water. Complete dissolution of precipitated clobetasol propionate was achieved by sonication for 30 minutes Sonorex RK 100H Bandelin Germany. The test solutions were freshly prepared one day prior the start of the experiment and were constantly aerated at room temperature. The glass wells for the incubation of the embryos were pre saturated with the respective test solution overnight and renewed at the beginning of the experiment. Per sample about 40 50 fertilized fish eggs were pre picked in glass petri dishes 10 cm diameter filled with 30 ml of the testing solution. Using an optical binokular microscope 20 embryos of the same early blastula stage were transferred from the pre picking plate into the test glass wells filled with 8 ml of the test solution. Embryos were then incubated as described in the growth protocol. Standard flagellin from Bacillus subtilis 10% purity Pam3CSK4 98 % purity and PolyI:C HMW high molecular weight were purchased from InvivoGen SAS Toulouse France. Stock solutions 1 mg/mL for polyI:C and Pam3CSK4; 0.1 mg/mL for flagellin were prepared in provided endotoxin free water aliquoted and stored according to the manufacturers instructions until further use. At approximately 46 hpf the embryos were manually dechorinated and then further incubated. A mixture of the different PAMPs was prepared by mixing 10 L of each component resulting in final concentrations of 0.33 mg/mL for polyI:C and Pam3CSK4 and 0.033 mg/mL for flagellin respectively. The device for microinjection was set up using FemtoTip II injection needles with a complementary universal capillary holder Eppendorf Hamburg Germany connected to an IM 300 microinjector Narishige London United Kingdom. Volume calibration was performed according to Sive et al. 2010 using a micrometer slide with 1/100 mm scale Bresser Rhede Germany. Briefly the needles were backfilled with 10 L PAMP mix or ultra pure water. Injections for volume calibration were made into a drop of immersion oil purred on the micrometer slide the output pressure was constantly kept at 10 psi while the time of injection was adjusted until an injected drop had a diameter of approximately 250 m. Assuming that the drop forms a perfect sphere this corresponds to a volume of about 8 nL. The procedure described below was performed for each of the five conditions in turn treating the three replicates simultaneously. In order to anaesthetize the embryos tricaine Sigma Aldrich Taufkirchen Germany stock solution 4 mg/mL in 21 mM Tris pH 9 was added to the petri dishes to a final concentration of 200 g/mL. The immobilized embryos were transferred onto a 1 % agarose plate which was divided into segments one for each replicate. Excess liquid was removed so that the embryos were kept in place by surface tension. Desiccation was prevented by adding liquid if needed. To ensure that the injections could be performed as quickly as possible the embryos were arranged in a row in a way that their dorsal sides pointed to the same direction. The puncture was made on the dorsal side of the yolk sac and the needle was advanced through the yolk until it reached the yolk sac circulation valley on the ventral side of the embryo where the injection was made directly into the bloodstream. post all replicates of a certain condition were processed the embryos were rinsed back into the corresponding dish containing renewed test solution. Total RNA was extracted using the NucleoSpin RNA/Protein kit from Macherey Nagel 740933.250 The pooled and snap frozen larvae were suspended in RP1 buffer and were transferred to a screw cap tubes filled with 0.3 g of Lysing Matrix D MP Biomedicals 6913050 ceramic beads. Tissue homogenization was performed at 5 m/s for 45 s with FastPrep 24 MP Biomedicals 6004500 at room temperature. From here on RNA was extracted according to the manufacturers protocol. RNA concentration >100 ng/l was measured using a Nanodrop 2000 spectrophotometer Thermo Scientific. The samples overall RNA quality and corresponding RIN value was fluorometrically determined with a 2100 Bioanalyzer Instrument system Agilent G2939BA using the Agilent RNA 6000 Nano Kit Agilent 5067 1511 according to the manufacturers protocol. Only samples with a RIN > 8 were used for downstream analysis. Samples were stored at 80C until they were send to the sequencing facility on dry ice. Sequencing libraries were prepared for each sample from 100 ng/l total RNA at the sequencing facility NGS Services for Integrative Genomics at the University of Gttingen in Germany. According to their standard workflow cDNA libraries were prepared from protein coding mRNA that were purified through PolyA selection using the TruSeq RNA Library Prep Kit v2 Illumina. Libraries were validated using a Fragment Analyzer system Agilent Santa Clara USA before sequencing.,Experimental Factor: compound:n1|Experimental Factor: stimulus:n1,RNA-Seq,TRANSCRIPTOMIC,PolyA,SINGLE,ILLUMINA,Illumina HiSeq 4000,,ERP132743,Illumina HiSeq 4000 sequencing; Transcriptomic profiling of clobetasol propionate induced immunosuppression in challenged zebrafish embryos,ENA FIRST PUBLIC:2022 02 25|ENA LAST UPDATE:2022 02 25|loader:fastq load.py,p946sR1271_sr.fastq.gz,fastq,1998122568.0,39583977.0,E MTAB 11092:R1271,0:50.48,A:518205942;C:480545742;G:459346718;T:536870486;N:3153680,50,,,,518205942,480545742,459346718,536870486,3153680,ERX6749452,ERS8138758,ERA6860152,Fraunhofer Attract Eco,Fraunhofer Attract Eco,1,0.94252,,0.10542,,0.67886,,0.47083,,35,,B,,usable mapping rate,illumina,hiseq_era,unknown,poly_a,trueseq,bulk,unknown,unknown,,Germany,2022-01-01,Hatching,Embryo,Whole Organism,All anatomical structures
10319,ERR7179894,ERX6749451,ERS8138757,ERP132743,PRJEB48372,Transcriptomic profiling of clobetasol propionate induced immunosuppression in challenged zebrafish embryos,E-MTAB-11092,Transcriptome Analysis,The aim of this experiment was to develop an approach for the assessment of immunotoxicity in zebrafish embryos Danio rerio using transcriptomic profiling. Clobetasol propionate a highly potent synthetic corticosteroid which is known to have immunosuppressive effects on vertebrates including zebrafish was used for continuous exposure from fertilization until extraction. As immunotoxicity can only be comprehensively assessed when the effect is observed during an acute infection the embryos were injected with a mixture of different pathogen associated molecular patterns PAMPs consisting of Pam3CSK4 synthetic lipopeptide polyI:C synthetic viral dsRNA and flagellin isolated from Bacillus subtilis. Functional gene expression analysis from samples obtained by different kinds and combinations of treatments in relation to appropriate controls is used to identify pathways that are upregulated by artificial infection and suppressed by treatment with clobetasol propionate. In a modified version of the zebrafish embryo toxicity test OECD 236 20 fertilized eggs were exposed to a sub lethal concentration 250 nM of clobetasol propionate for xxx hours under semi static conditions. The test comprised of three treatments with clobetasol propionate and two negative control NC groups and was performed in triplicates. At 46 hpf the eggs were manually dechorinated. At 48 hpf the embryos were immobilized by anaesthetization using 200 mg/mL tricaine. Using microinjection 8 nL of the mixed PAMPs 0.33 µg/mL Pam3CSK4 0.33 µg/mL polyI:C and 0.033 µg/mL flagellin were injected directly into the embryos bloodstream. One of each exposure groups was injected with PAMPs while another one was injected with ultrapure water to control for effects introduced by the injection process. One control group remained completely untreated. post injection the embryos were further incubated for 3 hours before 10 embryos were randomly picked for each sample and pooled for RNA and protein extraction with NucleoSpin RNA/Protein kit Macherey Nagel. RNA quality was assessed with a 2100 Bioanalyzer system Agilent before coding RNA was purified PolyA selection with TruSeq RNA Library Prep Kit v2 and sequenced on an Illumina HiSeq 4000 System Illumina in 50 bp single read mode. Adapter sequences were removed with trimmomatic and sequences were aligned to the D.rerio reference genome GRCz11 with STAR. Counting of feature mapped reads was performed through featureCounts. Library gene count tables were then merged to a single count matrix as input for differential gene expression analysis with DESeq2.,ENA FIRST PUBLIC:2022 02 25|ENA LAST UPDATE:2022 02 25,,Protocols: For each sample 10 zebrafish larvae were randomly picked from the glass well and carefully transferred to a 1.5 ml low binding Eppendorf tube. Once individuals were collected for all samples the supernatant was removed and the tubes were snap frozen in liquid nitrogen. For detailed information about embryonic incubation conditions and husbandry of adult broodstock please refer to the “Growth protocol”. For detailed information about exposure duration and conditions please refer to the “Treatment protocol”. Adult animal husbandry: Wild type zebrafish Danio rerio Strain AB broodstocks were maintained under flow through conditions in 150 L tanks at 26 +/ 2°C on a 12:12 h light/dark cycle. They were fed daily with TetraMin® Tetra Werke Melle Germany main feed ad libitum and nauplii of Artemia salina. The day before test start glass spawning trays with artificial substrate green glass beads stringed on stainless steel wire were placed at the bottom of each tank. post mating and spawning in the morning hours eggs were rinsed with clear Cu reduced water and placed into glass dishes for pre picking. Fish embryo incubation: For each sample 20 fertilized eggs in the early blastula stage were placed in glass petri dishes diameter 6 cm filled with 8 mL of the testing solution or control medium. Embryos were incubated at 27 +/ 1°C on a 14:10 h light/dark cycle. At 24 hpf eggs were inspected while half of the test solution was renewed. Clobetasol propionate CAS: 25122 46 7 was purchased from Sigma Aldrich Taufkirchen Germany. For the test solution preparation first a 5 mM stock solution was prepared in Acetone 99.9% MS grade. This stock was diluted to 250 µM in acetone before 500 µL were added to a 500 mL DURAN glass bottle. To the bottle for the control solution only 500 µL of pure Acetone was added. post complete evaporation of the acetone the bottle was filled up with 500 mL copper reduced tap water. Complete dissolution of precipitated clobetasol propionate was achieved by sonication for 30 minutes Sonorex RK 100H Bandelin Germany. The test solutions were freshly prepared one day prior the start of the experiment and were constantly aerated at room temperature. The glass wells for the incubation of the embryos were pre saturated with the respective test solution overnight and renewed at the beginning of the experiment. Per sample about 40 50 fertilized fish eggs were pre picked in glass petri dishes 10 cm diameter filled with 30 ml of the testing solution. Using an optical binokular microscope 20 embryos of the same early blastula stage were transferred from the pre picking plate into the test glass wells filled with 8 ml of the test solution. Embryos were then incubated as described in the “growth protocol”. Standard flagellin from Bacillus subtilis ≈ 10% purity Pam3CSK4 ≥ 98 % purity and PolyI:C HMW high molecular weight were purchased from InvivoGen SAS Toulouse France. Stock solutions 1 mg/mL for polyI:C and Pam3CSK4; 0.1 mg/mL for flagellin were prepared in provided endotoxin free water aliquoted and stored according to the manufacturer's instructions until further use. At approximately 46 hpf the embryos were manually dechorinated and then further incubated. A mixture of the different PAMPs was prepared by mixing 10 µL of each component resulting in final concentrations of 0.33 mg/mL for polyI:C and Pam3CSK4 and 0.033 mg/mL for flagellin respectively. The device for microinjection was set up using FemtoTip II injection needles with a complementary universal capillary holder Eppendorf Hamburg Germany connected to an IM 300 microinjector Narishige London United Kingdom. Volume calibration was performed according to Sive et al. 2010 using a micrometer slide with 1/100 mm scale Bresser Rhede Germany. Briefly the needles were backfilled with 10 µL PAMP mix or ultra pure water. Injections for volume calibration were made into a drop of immersion oil purred on the micrometer slide the output pressure was constantly kept at 10 psi while the time of injection was adjusted until an injected drop had a diameter of approximately 250 µm. Assuming that the drop forms a perfect sphere this corresponds to a volume of about 8 nL. The procedure described below was performed for each of the five conditions in turn treating the three replicates simultaneously. In order to anaesthetize the embryos tricaine Sigma Aldrich Taufkirchen Germany stock solution 4 mg/mL in 21 mM Tris pH 9 was added to the petri dishes to a final concentration of 200 µg/mL. The immobilized embryos were transferred onto a 1 % agarose plate which was divided into segments one for each replicate. Excess liquid was removed so that the embryos were kept in place by surface tension. Desiccation was prevented by adding liquid if needed. To ensure that the injections could be performed as quickly as possible the embryos were arranged in a row in a way that their dorsal sides pointed to the same direction. The puncture was made on the dorsal side of the yolk sac and the needle was advanced through the yolk until it reached the yolk sac circulation valley on the ventral side of the embryo where the injection was made directly into the bloodstream. post all replicates of a certain condition were processed the embryos were rinsed back into the corresponding dish containing renewed test solution. Total RNA was extracted using the NucleoSpin RNA/Protein kit from Macherey Nagel 740933.250 The pooled and snap frozen larvae were suspended in RP1 buffer and were transferred to a screw cap tubes filled with 0.3 g of Lysing Matrix D MP Biomedicals 6913050 ceramic beads. Tissue homogenization was performed at 5 m/s for 45 s with FastPrep 24© MP Biomedicals 6004500 at room temperature. From here on RNA was extracted according to the manufacturer's protocol. RNA concentration >100 ng/µl was measured using a Nanodrop 2000 spectrophotometer Thermo Scientific. The sample's overall RNA quality and corresponding RIN value was fluorometrically determined with a 2100 Bioanalyzer© Instrument system Agilent G2939BA using the Agilent RNA 6000 Nano Kit© Agilent 5067 1511 according to the manufacturer's protocol. Only samples with a RIN > 8 were used for downstream analysis. Samples were stored at 80°C until they were send to the sequencing facility on dry ice. Sequencing libraries were prepared for each sample from 100 ng/µl total RNA at the sequencing facility “NGS Services for Integrative Genomics” at the University of Göttingen in Germany. According to their standard workflow cDNA libraries were prepared from protein coding mRNA that were purified through PolyA selection using the TruSeq RNA Library Prep Kit v2 Illumina. Libraries were validated using a Fragment Analyzer system Agilent Santa Clara USA before sequencing.,R1288,SAMEA10486002,"Fraunhofer Attract Eco'n'OMICs, Fraunhofer Institute for Molecular Biology and Applied Ecology, Schmallenberg, Germany",ENA first public:2022 01 01|ENA last update:2022 01 01|External Id:SAMEA10486002|INSDC center alias:Fraunhofer Attract Eco'n'OMICs Fraunhofer Institute for Molecular Biology and Applied Ecology Schmallenberg Germany|INSDC center name:Fraunhofer Attract Eco'n'OMICs Fraunhofer Institute for Molecular Biology and Applied Ecology Schmallenberg Germany|INSDC first public:2022 01 01T00:13:13Z|INSDC last update:2022 01 01T00:13:13Z|INSDC status:public|Submitter Id:E MTAB 11092:R1288|age:51|broker name:ArrayExpress|common name:zebrafish|developmental stage:hatching long pec|genotype:wild type genotype|organism part:whole organism|replicate:T12|sample name:E MTAB 11092:R1288|sex:mixed|stimulus:PAMP|strain:AB,,,,,,,,,Illumina HiSeq 4000 sequencing; Transcriptomic profiling of clobetasol propionate induced immunosuppression in challenged zebrafish embryos,E MTAB 11092:R1288 s,R1288 s,Transcriptomic profiling of clobetasol propionate induced immunosuppression in challenged zebrafish embryos,For each sample 10 zebrafish larvae were randomly picked from the glass well and carefully transferred to a 1.5 ml low binding Eppendorf tube. Once individuals were collected for all samples the supernatant was removed and the tubes were snap frozen in liquid nitrogen. For detailed information about embryonic incubation conditions and husbandry of adult broodstock please refer to the Growth protocol. For detailed information about exposure duration and conditions please refer to the Treatment protocol. Adult animal husbandry: Wild type zebrafish Danio rerio Strain AB broodstocks were maintained under flow through conditions in 150 L tanks at 26 +/ 2C on a 12:12 h light/dark cycle. They were fed daily with TetraMin Tetra Werke Melle Germany main feed ad libitum and nauplii of Artemia salina. The day before test start glass spawning trays with artificial substrate green glass beads stringed on stainless steel wire were placed at the bottom of each tank. post mating and spawning in the morning hours eggs were rinsed with clear Cu reduced water and placed into glass dishes for pre picking. Fish embryo incubation: For each sample 20 fertilized eggs in the early blastula stage were placed in glass petri dishes diameter 6 cm filled with 8 mL of the testing solution or control medium. Embryos were incubated at 27 +/ 1C on a 14:10 h light/dark cycle. At 24 hpf eggs were inspected while half of the test solution was renewed. Clobetasol propionate CAS: 25122 46 7 was purchased from Sigma Aldrich Taufkirchen Germany. For the test solution preparation first a 5 mM stock solution was prepared in Acetone 99.9% MS grade. This stock was diluted to 250 M in acetone before 500 L were added to a 500 mL DURAN glass bottle. To the bottle for the control solution only 500 L of pure Acetone was added. post complete evaporation of the acetone the bottle was filled up with 500 mL copper reduced tap water. Complete dissolution of precipitated clobetasol propionate was achieved by sonication for 30 minutes Sonorex RK 100H Bandelin Germany. The test solutions were freshly prepared one day prior the start of the experiment and were constantly aerated at room temperature. The glass wells for the incubation of the embryos were pre saturated with the respective test solution overnight and renewed at the beginning of the experiment. Per sample about 40 50 fertilized fish eggs were pre picked in glass petri dishes 10 cm diameter filled with 30 ml of the testing solution. Using an optical binokular microscope 20 embryos of the same early blastula stage were transferred from the pre picking plate into the test glass wells filled with 8 ml of the test solution. Embryos were then incubated as described in the growth protocol. Standard flagellin from Bacillus subtilis 10% purity Pam3CSK4 98 % purity and PolyI:C HMW high molecular weight were purchased from InvivoGen SAS Toulouse France. Stock solutions 1 mg/mL for polyI:C and Pam3CSK4; 0.1 mg/mL for flagellin were prepared in provided endotoxin free water aliquoted and stored according to the manufacturers instructions until further use. At approximately 46 hpf the embryos were manually dechorinated and then further incubated. A mixture of the different PAMPs was prepared by mixing 10 L of each component resulting in final concentrations of 0.33 mg/mL for polyI:C and Pam3CSK4 and 0.033 mg/mL for flagellin respectively. The device for microinjection was set up using FemtoTip II injection needles with a complementary universal capillary holder Eppendorf Hamburg Germany connected to an IM 300 microinjector Narishige London United Kingdom. Volume calibration was performed according to Sive et al. 2010 using a micrometer slide with 1/100 mm scale Bresser Rhede Germany. Briefly the needles were backfilled with 10 L PAMP mix or ultra pure water. Injections for volume calibration were made into a drop of immersion oil purred on the micrometer slide the output pressure was constantly kept at 10 psi while the time of injection was adjusted until an injected drop had a diameter of approximately 250 m. Assuming that the drop forms a perfect sphere this corresponds to a volume of about 8 nL. The procedure described below was performed for each of the five conditions in turn treating the three replicates simultaneously. In order to anaesthetize the embryos tricaine Sigma Aldrich Taufkirchen Germany stock solution 4 mg/mL in 21 mM Tris pH 9 was added to the petri dishes to a final concentration of 200 g/mL. The immobilized embryos were transferred onto a 1 % agarose plate which was divided into segments one for each replicate. Excess liquid was removed so that the embryos were kept in place by surface tension. Desiccation was prevented by adding liquid if needed. To ensure that the injections could be performed as quickly as possible the embryos were arranged in a row in a way that their dorsal sides pointed to the same direction. The puncture was made on the dorsal side of the yolk sac and the needle was advanced through the yolk until it reached the yolk sac circulation valley on the ventral side of the embryo where the injection was made directly into the bloodstream. post all replicates of a certain condition were processed the embryos were rinsed back into the corresponding dish containing renewed test solution. Total RNA was extracted using the NucleoSpin RNA/Protein kit from Macherey Nagel 740933.250 The pooled and snap frozen larvae were suspended in RP1 buffer and were transferred to a screw cap tubes filled with 0.3 g of Lysing Matrix D MP Biomedicals 6913050 ceramic beads. Tissue homogenization was performed at 5 m/s for 45 s with FastPrep 24 MP Biomedicals 6004500 at room temperature. From here on RNA was extracted according to the manufacturers protocol. RNA concentration >100 ng/l was measured using a Nanodrop 2000 spectrophotometer Thermo Scientific. The samples overall RNA quality and corresponding RIN value was fluorometrically determined with a 2100 Bioanalyzer Instrument system Agilent G2939BA using the Agilent RNA 6000 Nano Kit Agilent 5067 1511 according to the manufacturers protocol. Only samples with a RIN > 8 were used for downstream analysis. Samples were stored at 80C until they were send to the sequencing facility on dry ice. Sequencing libraries were prepared for each sample from 100 ng/l total RNA at the sequencing facility NGS Services for Integrative Genomics at the University of Gttingen in Germany. According to their standard workflow cDNA libraries were prepared from protein coding mRNA that were purified through PolyA selection using the TruSeq RNA Library Prep Kit v2 Illumina. Libraries were validated using a Fragment Analyzer system Agilent Santa Clara USA before sequencing.,Experimental Factor: compound:Clobetasol propionate|Experimental Factor: dose:250|Experimental Factor: stimulus:PAMP,RNA-Seq,TRANSCRIPTOMIC,PolyA,SINGLE,ILLUMINA,Illumina HiSeq 4000,,ERP132743,Illumina HiSeq 4000 sequencing; Transcriptomic profiling of clobetasol propionate induced immunosuppression in challenged zebrafish embryos,ENA FIRST PUBLIC:2022 02 25|ENA LAST UPDATE:2022 02 25|loader:fastq load.py,p946sR1288_sr.fastq.gz,fastq,1892621026.0,37517169.0,E MTAB 11092:R1288,0:50.45,A:498344488;C:444226431;G:428956415;T:514938257;N:6155435,50,,,,498344488,444226431,428956415,514938257,6155435,ERX6749451,ERS8138757,ERA6860152,Fraunhofer Attract Eco,Fraunhofer Attract Eco,1,0.93409,,0.12599,,0.68692,,0.47741,,51,,B,,usable mapping rate,illumina,hiseq_era,unknown,poly_a,trueseq,bulk,unknown,unknown,,Germany,2022-01-01,Hatching,Embryo,Whole Organism,All anatomical structures
10320,ERR7179893,ERX6749450,ERS8138756,ERP132743,PRJEB48372,Transcriptomic profiling of clobetasol propionate induced immunosuppression in challenged zebrafish embryos,E-MTAB-11092,Transcriptome Analysis,The aim of this experiment was to develop an approach for the assessment of immunotoxicity in zebrafish embryos Danio rerio using transcriptomic profiling. Clobetasol propionate a highly potent synthetic corticosteroid which is known to have immunosuppressive effects on vertebrates including zebrafish was used for continuous exposure from fertilization until extraction. As immunotoxicity can only be comprehensively assessed when the effect is observed during an acute infection the embryos were injected with a mixture of different pathogen associated molecular patterns PAMPs consisting of Pam3CSK4 synthetic lipopeptide polyI:C synthetic viral dsRNA and flagellin isolated from Bacillus subtilis. Functional gene expression analysis from samples obtained by different kinds and combinations of treatments in relation to appropriate controls is used to identify pathways that are upregulated by artificial infection and suppressed by treatment with clobetasol propionate. In a modified version of the zebrafish embryo toxicity test OECD 236 20 fertilized eggs were exposed to a sub lethal concentration 250 nM of clobetasol propionate for xxx hours under semi static conditions. The test comprised of three treatments with clobetasol propionate and two negative control NC groups and was performed in triplicates. At 46 hpf the eggs were manually dechorinated. At 48 hpf the embryos were immobilized by anaesthetization using 200 mg/mL tricaine. Using microinjection 8 nL of the mixed PAMPs 0.33 µg/mL Pam3CSK4 0.33 µg/mL polyI:C and 0.033 µg/mL flagellin were injected directly into the embryos bloodstream. One of each exposure groups was injected with PAMPs while another one was injected with ultrapure water to control for effects introduced by the injection process. One control group remained completely untreated. post injection the embryos were further incubated for 3 hours before 10 embryos were randomly picked for each sample and pooled for RNA and protein extraction with NucleoSpin RNA/Protein kit Macherey Nagel. RNA quality was assessed with a 2100 Bioanalyzer system Agilent before coding RNA was purified PolyA selection with TruSeq RNA Library Prep Kit v2 and sequenced on an Illumina HiSeq 4000 System Illumina in 50 bp single read mode. Adapter sequences were removed with trimmomatic and sequences were aligned to the D.rerio reference genome GRCz11 with STAR. Counting of feature mapped reads was performed through featureCounts. Library gene count tables were then merged to a single count matrix as input for differential gene expression analysis with DESeq2.,ENA FIRST PUBLIC:2022 02 25|ENA LAST UPDATE:2022 02 25,,Protocols: For each sample 10 zebrafish larvae were randomly picked from the glass well and carefully transferred to a 1.5 ml low binding Eppendorf tube. Once individuals were collected for all samples the supernatant was removed and the tubes were snap frozen in liquid nitrogen. For detailed information about embryonic incubation conditions and husbandry of adult broodstock please refer to the “Growth protocol”. For detailed information about exposure duration and conditions please refer to the “Treatment protocol”. Adult animal husbandry: Wild type zebrafish Danio rerio Strain AB broodstocks were maintained under flow through conditions in 150 L tanks at 26 +/ 2°C on a 12:12 h light/dark cycle. They were fed daily with TetraMin® Tetra Werke Melle Germany main feed ad libitum and nauplii of Artemia salina. The day before test start glass spawning trays with artificial substrate green glass beads stringed on stainless steel wire were placed at the bottom of each tank. post mating and spawning in the morning hours eggs were rinsed with clear Cu reduced water and placed into glass dishes for pre picking. Fish embryo incubation: For each sample 20 fertilized eggs in the early blastula stage were placed in glass petri dishes diameter 6 cm filled with 8 mL of the testing solution or control medium. Embryos were incubated at 27 +/ 1°C on a 14:10 h light/dark cycle. At 24 hpf eggs were inspected while half of the test solution was renewed. Clobetasol propionate CAS: 25122 46 7 was purchased from Sigma Aldrich Taufkirchen Germany. For the test solution preparation first a 5 mM stock solution was prepared in Acetone 99.9% MS grade. This stock was diluted to 250 µM in acetone before 500 µL were added to a 500 mL DURAN glass bottle. To the bottle for the control solution only 500 µL of pure Acetone was added. post complete evaporation of the acetone the bottle was filled up with 500 mL copper reduced tap water. Complete dissolution of precipitated clobetasol propionate was achieved by sonication for 30 minutes Sonorex RK 100H Bandelin Germany. The test solutions were freshly prepared one day prior the start of the experiment and were constantly aerated at room temperature. The glass wells for the incubation of the embryos were pre saturated with the respective test solution overnight and renewed at the beginning of the experiment. Per sample about 40 50 fertilized fish eggs were pre picked in glass petri dishes 10 cm diameter filled with 30 ml of the testing solution. Using an optical binokular microscope 20 embryos of the same early blastula stage were transferred from the pre picking plate into the test glass wells filled with 8 ml of the test solution. Embryos were then incubated as described in the “growth protocol”. Standard flagellin from Bacillus subtilis ≈ 10% purity Pam3CSK4 ≥ 98 % purity and PolyI:C HMW high molecular weight were purchased from InvivoGen SAS Toulouse France. Stock solutions 1 mg/mL for polyI:C and Pam3CSK4; 0.1 mg/mL for flagellin were prepared in provided endotoxin free water aliquoted and stored according to the manufacturer's instructions until further use. At approximately 46 hpf the embryos were manually dechorinated and then further incubated. A mixture of the different PAMPs was prepared by mixing 10 µL of each component resulting in final concentrations of 0.33 mg/mL for polyI:C and Pam3CSK4 and 0.033 mg/mL for flagellin respectively. The device for microinjection was set up using FemtoTip II injection needles with a complementary universal capillary holder Eppendorf Hamburg Germany connected to an IM 300 microinjector Narishige London United Kingdom. Volume calibration was performed according to Sive et al. 2010 using a micrometer slide with 1/100 mm scale Bresser Rhede Germany. Briefly the needles were backfilled with 10 µL PAMP mix or ultra pure water. Injections for volume calibration were made into a drop of immersion oil purred on the micrometer slide the output pressure was constantly kept at 10 psi while the time of injection was adjusted until an injected drop had a diameter of approximately 250 µm. Assuming that the drop forms a perfect sphere this corresponds to a volume of about 8 nL. The procedure described below was performed for each of the five conditions in turn treating the three replicates simultaneously. In order to anaesthetize the embryos tricaine Sigma Aldrich Taufkirchen Germany stock solution 4 mg/mL in 21 mM Tris pH 9 was added to the petri dishes to a final concentration of 200 µg/mL. The immobilized embryos were transferred onto a 1 % agarose plate which was divided into segments one for each replicate. Excess liquid was removed so that the embryos were kept in place by surface tension. Desiccation was prevented by adding liquid if needed. To ensure that the injections could be performed as quickly as possible the embryos were arranged in a row in a way that their dorsal sides pointed to the same direction. The puncture was made on the dorsal side of the yolk sac and the needle was advanced through the yolk until it reached the yolk sac circulation valley on the ventral side of the embryo where the injection was made directly into the bloodstream. post all replicates of a certain condition were processed the embryos were rinsed back into the corresponding dish containing renewed test solution. Total RNA was extracted using the NucleoSpin RNA/Protein kit from Macherey Nagel 740933.250 The pooled and snap frozen larvae were suspended in RP1 buffer and were transferred to a screw cap tubes filled with 0.3 g of Lysing Matrix D MP Biomedicals 6913050 ceramic beads. Tissue homogenization was performed at 5 m/s for 45 s with FastPrep 24© MP Biomedicals 6004500 at room temperature. From here on RNA was extracted according to the manufacturer's protocol. RNA concentration >100 ng/µl was measured using a Nanodrop 2000 spectrophotometer Thermo Scientific. The sample's overall RNA quality and corresponding RIN value was fluorometrically determined with a 2100 Bioanalyzer© Instrument system Agilent G2939BA using the Agilent RNA 6000 Nano Kit© Agilent 5067 1511 according to the manufacturer's protocol. Only samples with a RIN > 8 were used for downstream analysis. Samples were stored at 80°C until they were send to the sequencing facility on dry ice. Sequencing libraries were prepared for each sample from 100 ng/µl total RNA at the sequencing facility “NGS Services for Integrative Genomics” at the University of Göttingen in Germany. According to their standard workflow cDNA libraries were prepared from protein coding mRNA that were purified through PolyA selection using the TruSeq RNA Library Prep Kit v2 Illumina. Libraries were validated using a Fragment Analyzer system Agilent Santa Clara USA before sequencing.,R1285,SAMEA10486000,"Fraunhofer Attract Eco'n'OMICs, Fraunhofer Institute for Molecular Biology and Applied Ecology, Schmallenberg, Germany",ENA first public:2022 01 01|ENA last update:2022 01 01|External Id:SAMEA10486000|INSDC center alias:Fraunhofer Attract Eco'n'OMICs Fraunhofer Institute for Molecular Biology and Applied Ecology Schmallenberg Germany|INSDC center name:Fraunhofer Attract Eco'n'OMICs Fraunhofer Institute for Molecular Biology and Applied Ecology Schmallenberg Germany|INSDC first public:2022 01 01T00:13:13Z|INSDC last update:2022 01 01T00:13:13Z|INSDC status:public|Submitter Id:E MTAB 11092:R1285|age:51|broker name:ArrayExpress|common name:zebrafish|developmental stage:hatching long pec|genotype:wild type genotype|organism part:whole organism|replicate:T12|sample name:E MTAB 11092:R1285|sex:mixed|stimulus:water|strain:AB,,,,,,,,,Illumina HiSeq 4000 sequencing; Transcriptomic profiling of clobetasol propionate induced immunosuppression in challenged zebrafish embryos,E MTAB 11092:R1285 s,R1285 s,Transcriptomic profiling of clobetasol propionate induced immunosuppression in challenged zebrafish embryos,For each sample 10 zebrafish larvae were randomly picked from the glass well and carefully transferred to a 1.5 ml low binding Eppendorf tube. Once individuals were collected for all samples the supernatant was removed and the tubes were snap frozen in liquid nitrogen. For detailed information about embryonic incubation conditions and husbandry of adult broodstock please refer to the Growth protocol. For detailed information about exposure duration and conditions please refer to the Treatment protocol. Adult animal husbandry: Wild type zebrafish Danio rerio Strain AB broodstocks were maintained under flow through conditions in 150 L tanks at 26 +/ 2C on a 12:12 h light/dark cycle. They were fed daily with TetraMin Tetra Werke Melle Germany main feed ad libitum and nauplii of Artemia salina. The day before test start glass spawning trays with artificial substrate green glass beads stringed on stainless steel wire were placed at the bottom of each tank. post mating and spawning in the morning hours eggs were rinsed with clear Cu reduced water and placed into glass dishes for pre picking. Fish embryo incubation: For each sample 20 fertilized eggs in the early blastula stage were placed in glass petri dishes diameter 6 cm filled with 8 mL of the testing solution or control medium. Embryos were incubated at 27 +/ 1C on a 14:10 h light/dark cycle. At 24 hpf eggs were inspected while half of the test solution was renewed. Clobetasol propionate CAS: 25122 46 7 was purchased from Sigma Aldrich Taufkirchen Germany. For the test solution preparation first a 5 mM stock solution was prepared in Acetone 99.9% MS grade. This stock was diluted to 250 M in acetone before 500 L were added to a 500 mL DURAN glass bottle. To the bottle for the control solution only 500 L of pure Acetone was added. post complete evaporation of the acetone the bottle was filled up with 500 mL copper reduced tap water. Complete dissolution of precipitated clobetasol propionate was achieved by sonication for 30 minutes Sonorex RK 100H Bandelin Germany. The test solutions were freshly prepared one day prior the start of the experiment and were constantly aerated at room temperature. The glass wells for the incubation of the embryos were pre saturated with the respective test solution overnight and renewed at the beginning of the experiment. Per sample about 40 50 fertilized fish eggs were pre picked in glass petri dishes 10 cm diameter filled with 30 ml of the testing solution. Using an optical binokular microscope 20 embryos of the same early blastula stage were transferred from the pre picking plate into the test glass wells filled with 8 ml of the test solution. Embryos were then incubated as described in the growth protocol. Standard flagellin from Bacillus subtilis 10% purity Pam3CSK4 98 % purity and PolyI:C HMW high molecular weight were purchased from InvivoGen SAS Toulouse France. Stock solutions 1 mg/mL for polyI:C and Pam3CSK4; 0.1 mg/mL for flagellin were prepared in provided endotoxin free water aliquoted and stored according to the manufacturers instructions until further use. At approximately 46 hpf the embryos were manually dechorinated and then further incubated. A mixture of the different PAMPs was prepared by mixing 10 L of each component resulting in final concentrations of 0.33 mg/mL for polyI:C and Pam3CSK4 and 0.033 mg/mL for flagellin respectively. The device for microinjection was set up using FemtoTip II injection needles with a complementary universal capillary holder Eppendorf Hamburg Germany connected to an IM 300 microinjector Narishige London United Kingdom. Volume calibration was performed according to Sive et al. 2010 using a micrometer slide with 1/100 mm scale Bresser Rhede Germany. Briefly the needles were backfilled with 10 L PAMP mix or ultra pure water. Injections for volume calibration were made into a drop of immersion oil purred on the micrometer slide the output pressure was constantly kept at 10 psi while the time of injection was adjusted until an injected drop had a diameter of approximately 250 m. Assuming that the drop forms a perfect sphere this corresponds to a volume of about 8 nL. The procedure described below was performed for each of the five conditions in turn treating the three replicates simultaneously. In order to anaesthetize the embryos tricaine Sigma Aldrich Taufkirchen Germany stock solution 4 mg/mL in 21 mM Tris pH 9 was added to the petri dishes to a final concentration of 200 g/mL. The immobilized embryos were transferred onto a 1 % agarose plate which was divided into segments one for each replicate. Excess liquid was removed so that the embryos were kept in place by surface tension. Desiccation was prevented by adding liquid if needed. To ensure that the injections could be performed as quickly as possible the embryos were arranged in a row in a way that their dorsal sides pointed to the same direction. The puncture was made on the dorsal side of the yolk sac and the needle was advanced through the yolk until it reached the yolk sac circulation valley on the ventral side of the embryo where the injection was made directly into the bloodstream. post all replicates of a certain condition were processed the embryos were rinsed back into the corresponding dish containing renewed test solution. Total RNA was extracted using the NucleoSpin RNA/Protein kit from Macherey Nagel 740933.250 The pooled and snap frozen larvae were suspended in RP1 buffer and were transferred to a screw cap tubes filled with 0.3 g of Lysing Matrix D MP Biomedicals 6913050 ceramic beads. Tissue homogenization was performed at 5 m/s for 45 s with FastPrep 24 MP Biomedicals 6004500 at room temperature. From here on RNA was extracted according to the manufacturers protocol. RNA concentration >100 ng/l was measured using a Nanodrop 2000 spectrophotometer Thermo Scientific. The samples overall RNA quality and corresponding RIN value was fluorometrically determined with a 2100 Bioanalyzer Instrument system Agilent G2939BA using the Agilent RNA 6000 Nano Kit Agilent 5067 1511 according to the manufacturers protocol. Only samples with a RIN > 8 were used for downstream analysis. Samples were stored at 80C until they were send to the sequencing facility on dry ice. Sequencing libraries were prepared for each sample from 100 ng/l total RNA at the sequencing facility NGS Services for Integrative Genomics at the University of Gttingen in Germany. According to their standard workflow cDNA libraries were prepared from protein coding mRNA that were purified through PolyA selection using the TruSeq RNA Library Prep Kit v2 Illumina. Libraries were validated using a Fragment Analyzer system Agilent Santa Clara USA before sequencing.,Experimental Factor: compound:Clobetasol propionate|Experimental Factor: dose:250|Experimental Factor: stimulus:water,RNA-Seq,TRANSCRIPTOMIC,PolyA,SINGLE,ILLUMINA,Illumina HiSeq 4000,,ERP132743,Illumina HiSeq 4000 sequencing; Transcriptomic profiling of clobetasol propionate induced immunosuppression in challenged zebrafish embryos,ENA FIRST PUBLIC:2022 02 25|ENA LAST UPDATE:2022 02 25|loader:fastq load.py,p946sR1285_sr.fastq.gz,fastq,1829128750.0,36225046.0,E MTAB 11092:R1285,0:50.49,A:478165041;C:438683413;G:419515205;T:490751870;N:2013221,50,,,,478165041,438683413,419515205,490751870,2013221,ERX6749450,ERS8138756,ERA6860152,Fraunhofer Attract Eco,Fraunhofer Attract Eco,1,0.93814,,0.11901,,0.68639,,0.46045,,50,,B,,usable mapping rate,illumina,hiseq_era,unknown,poly_a,trueseq,bulk,unknown,unknown,,Germany,2022-01-01,Hatching,Embryo,Whole Organism,All anatomical structures
10321,ERR7179892,ERX6749449,ERS8138755,ERP132743,PRJEB48372,Transcriptomic profiling of clobetasol propionate induced immunosuppression in challenged zebrafish embryos,E-MTAB-11092,Transcriptome Analysis,The aim of this experiment was to develop an approach for the assessment of immunotoxicity in zebrafish embryos Danio rerio using transcriptomic profiling. Clobetasol propionate a highly potent synthetic corticosteroid which is known to have immunosuppressive effects on vertebrates including zebrafish was used for continuous exposure from fertilization until extraction. As immunotoxicity can only be comprehensively assessed when the effect is observed during an acute infection the embryos were injected with a mixture of different pathogen associated molecular patterns PAMPs consisting of Pam3CSK4 synthetic lipopeptide polyI:C synthetic viral dsRNA and flagellin isolated from Bacillus subtilis. Functional gene expression analysis from samples obtained by different kinds and combinations of treatments in relation to appropriate controls is used to identify pathways that are upregulated by artificial infection and suppressed by treatment with clobetasol propionate. In a modified version of the zebrafish embryo toxicity test OECD 236 20 fertilized eggs were exposed to a sub lethal concentration 250 nM of clobetasol propionate for xxx hours under semi static conditions. The test comprised of three treatments with clobetasol propionate and two negative control NC groups and was performed in triplicates. At 46 hpf the eggs were manually dechorinated. At 48 hpf the embryos were immobilized by anaesthetization using 200 mg/mL tricaine. Using microinjection 8 nL of the mixed PAMPs 0.33 µg/mL Pam3CSK4 0.33 µg/mL polyI:C and 0.033 µg/mL flagellin were injected directly into the embryos bloodstream. One of each exposure groups was injected with PAMPs while another one was injected with ultrapure water to control for effects introduced by the injection process. One control group remained completely untreated. post injection the embryos were further incubated for 3 hours before 10 embryos were randomly picked for each sample and pooled for RNA and protein extraction with NucleoSpin RNA/Protein kit Macherey Nagel. RNA quality was assessed with a 2100 Bioanalyzer system Agilent before coding RNA was purified PolyA selection with TruSeq RNA Library Prep Kit v2 and sequenced on an Illumina HiSeq 4000 System Illumina in 50 bp single read mode. Adapter sequences were removed with trimmomatic and sequences were aligned to the D.rerio reference genome GRCz11 with STAR. Counting of feature mapped reads was performed through featureCounts. Library gene count tables were then merged to a single count matrix as input for differential gene expression analysis with DESeq2.,ENA FIRST PUBLIC:2022 02 25|ENA LAST UPDATE:2022 02 25,,Protocols: For each sample 10 zebrafish larvae were randomly picked from the glass well and carefully transferred to a 1.5 ml low binding Eppendorf tube. Once individuals were collected for all samples the supernatant was removed and the tubes were snap frozen in liquid nitrogen. For detailed information about embryonic incubation conditions and husbandry of adult broodstock please refer to the “Growth protocol”. For detailed information about exposure duration and conditions please refer to the “Treatment protocol”. Adult animal husbandry: Wild type zebrafish Danio rerio Strain AB broodstocks were maintained under flow through conditions in 150 L tanks at 26 +/ 2°C on a 12:12 h light/dark cycle. They were fed daily with TetraMin® Tetra Werke Melle Germany main feed ad libitum and nauplii of Artemia salina. The day before test start glass spawning trays with artificial substrate green glass beads stringed on stainless steel wire were placed at the bottom of each tank. post mating and spawning in the morning hours eggs were rinsed with clear Cu reduced water and placed into glass dishes for pre picking. Fish embryo incubation: For each sample 20 fertilized eggs in the early blastula stage were placed in glass petri dishes diameter 6 cm filled with 8 mL of the testing solution or control medium. Embryos were incubated at 27 +/ 1°C on a 14:10 h light/dark cycle. At 24 hpf eggs were inspected while half of the test solution was renewed. Clobetasol propionate CAS: 25122 46 7 was purchased from Sigma Aldrich Taufkirchen Germany. For the test solution preparation first a 5 mM stock solution was prepared in Acetone 99.9% MS grade. This stock was diluted to 250 µM in acetone before 500 µL were added to a 500 mL DURAN glass bottle. To the bottle for the control solution only 500 µL of pure Acetone was added. post complete evaporation of the acetone the bottle was filled up with 500 mL copper reduced tap water. Complete dissolution of precipitated clobetasol propionate was achieved by sonication for 30 minutes Sonorex RK 100H Bandelin Germany. The test solutions were freshly prepared one day prior the start of the experiment and were constantly aerated at room temperature. The glass wells for the incubation of the embryos were pre saturated with the respective test solution overnight and renewed at the beginning of the experiment. Per sample about 40 50 fertilized fish eggs were pre picked in glass petri dishes 10 cm diameter filled with 30 ml of the testing solution. Using an optical binokular microscope 20 embryos of the same early blastula stage were transferred from the pre picking plate into the test glass wells filled with 8 ml of the test solution. Embryos were then incubated as described in the “growth protocol”. Standard flagellin from Bacillus subtilis ≈ 10% purity Pam3CSK4 ≥ 98 % purity and PolyI:C HMW high molecular weight were purchased from InvivoGen SAS Toulouse France. Stock solutions 1 mg/mL for polyI:C and Pam3CSK4; 0.1 mg/mL for flagellin were prepared in provided endotoxin free water aliquoted and stored according to the manufacturer's instructions until further use. At approximately 46 hpf the embryos were manually dechorinated and then further incubated. A mixture of the different PAMPs was prepared by mixing 10 µL of each component resulting in final concentrations of 0.33 mg/mL for polyI:C and Pam3CSK4 and 0.033 mg/mL for flagellin respectively. The device for microinjection was set up using FemtoTip II injection needles with a complementary universal capillary holder Eppendorf Hamburg Germany connected to an IM 300 microinjector Narishige London United Kingdom. Volume calibration was performed according to Sive et al. 2010 using a micrometer slide with 1/100 mm scale Bresser Rhede Germany. Briefly the needles were backfilled with 10 µL PAMP mix or ultra pure water. Injections for volume calibration were made into a drop of immersion oil purred on the micrometer slide the output pressure was constantly kept at 10 psi while the time of injection was adjusted until an injected drop had a diameter of approximately 250 µm. Assuming that the drop forms a perfect sphere this corresponds to a volume of about 8 nL. The procedure described below was performed for each of the five conditions in turn treating the three replicates simultaneously. In order to anaesthetize the embryos tricaine Sigma Aldrich Taufkirchen Germany stock solution 4 mg/mL in 21 mM Tris pH 9 was added to the petri dishes to a final concentration of 200 µg/mL. The immobilized embryos were transferred onto a 1 % agarose plate which was divided into segments one for each replicate. Excess liquid was removed so that the embryos were kept in place by surface tension. Desiccation was prevented by adding liquid if needed. To ensure that the injections could be performed as quickly as possible the embryos were arranged in a row in a way that their dorsal sides pointed to the same direction. The puncture was made on the dorsal side of the yolk sac and the needle was advanced through the yolk until it reached the yolk sac circulation valley on the ventral side of the embryo where the injection was made directly into the bloodstream. post all replicates of a certain condition were processed the embryos were rinsed back into the corresponding dish containing renewed test solution. Total RNA was extracted using the NucleoSpin RNA/Protein kit from Macherey Nagel 740933.250 The pooled and snap frozen larvae were suspended in RP1 buffer and were transferred to a screw cap tubes filled with 0.3 g of Lysing Matrix D MP Biomedicals 6913050 ceramic beads. Tissue homogenization was performed at 5 m/s for 45 s with FastPrep 24© MP Biomedicals 6004500 at room temperature. From here on RNA was extracted according to the manufacturer's protocol. RNA concentration >100 ng/µl was measured using a Nanodrop 2000 spectrophotometer Thermo Scientific. The sample's overall RNA quality and corresponding RIN value was fluorometrically determined with a 2100 Bioanalyzer© Instrument system Agilent G2939BA using the Agilent RNA 6000 Nano Kit© Agilent 5067 1511 according to the manufacturer's protocol. Only samples with a RIN > 8 were used for downstream analysis. Samples were stored at 80°C until they were send to the sequencing facility on dry ice. Sequencing libraries were prepared for each sample from 100 ng/µl total RNA at the sequencing facility “NGS Services for Integrative Genomics” at the University of Göttingen in Germany. According to their standard workflow cDNA libraries were prepared from protein coding mRNA that were purified through PolyA selection using the TruSeq RNA Library Prep Kit v2 Illumina. Libraries were validated using a Fragment Analyzer system Agilent Santa Clara USA before sequencing.,R1276,SAMEA10485998,"Fraunhofer Attract Eco'n'OMICs, Fraunhofer Institute for Molecular Biology and Applied Ecology, Schmallenberg, Germany",ENA first public:2022 01 01|ENA last update:2022 01 01|External Id:SAMEA10485998|INSDC center alias:Fraunhofer Attract Eco'n'OMICs Fraunhofer Institute for Molecular Biology and Applied Ecology Schmallenberg Germany|INSDC center name:Fraunhofer Attract Eco'n'OMICs Fraunhofer Institute for Molecular Biology and Applied Ecology Schmallenberg Germany|INSDC first public:2022 01 01T00:13:13Z|INSDC last update:2022 01 01T00:13:13Z|INSDC status:public|Submitter Id:E MTAB 11092:R1276|age:51|broker name:ArrayExpress|common name:zebrafish|developmental stage:hatching long pec|genotype:wild type genotype|organism part:whole organism|replicate:T12|sample name:E MTAB 11092:R1276|sex:mixed|stimulus:PAMP|strain:AB,,,,,,,,,Illumina HiSeq 4000 sequencing; Transcriptomic profiling of clobetasol propionate induced immunosuppression in challenged zebrafish embryos,E MTAB 11092:R1276 s,R1276 s,Transcriptomic profiling of clobetasol propionate induced immunosuppression in challenged zebrafish embryos,For each sample 10 zebrafish larvae were randomly picked from the glass well and carefully transferred to a 1.5 ml low binding Eppendorf tube. Once individuals were collected for all samples the supernatant was removed and the tubes were snap frozen in liquid nitrogen. For detailed information about embryonic incubation conditions and husbandry of adult broodstock please refer to the Growth protocol. For detailed information about exposure duration and conditions please refer to the Treatment protocol. Adult animal husbandry: Wild type zebrafish Danio rerio Strain AB broodstocks were maintained under flow through conditions in 150 L tanks at 26 +/ 2C on a 12:12 h light/dark cycle. They were fed daily with TetraMin Tetra Werke Melle Germany main feed ad libitum and nauplii of Artemia salina. The day before test start glass spawning trays with artificial substrate green glass beads stringed on stainless steel wire were placed at the bottom of each tank. post mating and spawning in the morning hours eggs were rinsed with clear Cu reduced water and placed into glass dishes for pre picking. Fish embryo incubation: For each sample 20 fertilized eggs in the early blastula stage were placed in glass petri dishes diameter 6 cm filled with 8 mL of the testing solution or control medium. Embryos were incubated at 27 +/ 1C on a 14:10 h light/dark cycle. At 24 hpf eggs were inspected while half of the test solution was renewed. Clobetasol propionate CAS: 25122 46 7 was purchased from Sigma Aldrich Taufkirchen Germany. For the test solution preparation first a 5 mM stock solution was prepared in Acetone 99.9% MS grade. This stock was diluted to 250 M in acetone before 500 L were added to a 500 mL DURAN glass bottle. To the bottle for the control solution only 500 L of pure Acetone was added. post complete evaporation of the acetone the bottle was filled up with 500 mL copper reduced tap water. Complete dissolution of precipitated clobetasol propionate was achieved by sonication for 30 minutes Sonorex RK 100H Bandelin Germany. The test solutions were freshly prepared one day prior the start of the experiment and were constantly aerated at room temperature. The glass wells for the incubation of the embryos were pre saturated with the respective test solution overnight and renewed at the beginning of the experiment. Per sample about 40 50 fertilized fish eggs were pre picked in glass petri dishes 10 cm diameter filled with 30 ml of the testing solution. Using an optical binokular microscope 20 embryos of the same early blastula stage were transferred from the pre picking plate into the test glass wells filled with 8 ml of the test solution. Embryos were then incubated as described in the growth protocol. Standard flagellin from Bacillus subtilis 10% purity Pam3CSK4 98 % purity and PolyI:C HMW high molecular weight were purchased from InvivoGen SAS Toulouse France. Stock solutions 1 mg/mL for polyI:C and Pam3CSK4; 0.1 mg/mL for flagellin were prepared in provided endotoxin free water aliquoted and stored according to the manufacturers instructions until further use. At approximately 46 hpf the embryos were manually dechorinated and then further incubated. A mixture of the different PAMPs was prepared by mixing 10 L of each component resulting in final concentrations of 0.33 mg/mL for polyI:C and Pam3CSK4 and 0.033 mg/mL for flagellin respectively. The device for microinjection was set up using FemtoTip II injection needles with a complementary universal capillary holder Eppendorf Hamburg Germany connected to an IM 300 microinjector Narishige London United Kingdom. Volume calibration was performed according to Sive et al. 2010 using a micrometer slide with 1/100 mm scale Bresser Rhede Germany. Briefly the needles were backfilled with 10 L PAMP mix or ultra pure water. Injections for volume calibration were made into a drop of immersion oil purred on the micrometer slide the output pressure was constantly kept at 10 psi while the time of injection was adjusted until an injected drop had a diameter of approximately 250 m. Assuming that the drop forms a perfect sphere this corresponds to a volume of about 8 nL. The procedure described below was performed for each of the five conditions in turn treating the three replicates simultaneously. In order to anaesthetize the embryos tricaine Sigma Aldrich Taufkirchen Germany stock solution 4 mg/mL in 21 mM Tris pH 9 was added to the petri dishes to a final concentration of 200 g/mL. The immobilized embryos were transferred onto a 1 % agarose plate which was divided into segments one for each replicate. Excess liquid was removed so that the embryos were kept in place by surface tension. Desiccation was prevented by adding liquid if needed. To ensure that the injections could be performed as quickly as possible the embryos were arranged in a row in a way that their dorsal sides pointed to the same direction. The puncture was made on the dorsal side of the yolk sac and the needle was advanced through the yolk until it reached the yolk sac circulation valley on the ventral side of the embryo where the injection was made directly into the bloodstream. post all replicates of a certain condition were processed the embryos were rinsed back into the corresponding dish containing renewed test solution. Total RNA was extracted using the NucleoSpin RNA/Protein kit from Macherey Nagel 740933.250 The pooled and snap frozen larvae were suspended in RP1 buffer and were transferred to a screw cap tubes filled with 0.3 g of Lysing Matrix D MP Biomedicals 6913050 ceramic beads. Tissue homogenization was performed at 5 m/s for 45 s with FastPrep 24 MP Biomedicals 6004500 at room temperature. From here on RNA was extracted according to the manufacturers protocol. RNA concentration >100 ng/l was measured using a Nanodrop 2000 spectrophotometer Thermo Scientific. The samples overall RNA quality and corresponding RIN value was fluorometrically determined with a 2100 Bioanalyzer Instrument system Agilent G2939BA using the Agilent RNA 6000 Nano Kit Agilent 5067 1511 according to the manufacturers protocol. Only samples with a RIN > 8 were used for downstream analysis. Samples were stored at 80C until they were send to the sequencing facility on dry ice. Sequencing libraries were prepared for each sample from 100 ng/l total RNA at the sequencing facility NGS Services for Integrative Genomics at the University of Gttingen in Germany. According to their standard workflow cDNA libraries were prepared from protein coding mRNA that were purified through PolyA selection using the TruSeq RNA Library Prep Kit v2 Illumina. Libraries were validated using a Fragment Analyzer system Agilent Santa Clara USA before sequencing.,Experimental Factor: compound:n1|Experimental Factor: stimulus:PAMP,RNA-Seq,TRANSCRIPTOMIC,PolyA,SINGLE,ILLUMINA,Illumina HiSeq 4000,,ERP132743,Illumina HiSeq 4000 sequencing; Transcriptomic profiling of clobetasol propionate induced immunosuppression in challenged zebrafish embryos,ENA FIRST PUBLIC:2022 02 25|ENA LAST UPDATE:2022 02 25|loader:fastq load.py,p946sR1276_sr.fastq.gz,fastq,1575013709.0,31202630.0,E MTAB 11092:R1276,0:50.48,A:406866583;C:380336879;G:364368178;T:420970851;N:2471218,50,,,,406866583,380336879,364368178,420970851,2471218,ERX6749449,ERS8138755,ERA6860152,Fraunhofer Attract Eco,Fraunhofer Attract Eco,1,0.9399,,0.10316,,0.68564,,0.45479,,49,,B,,usable mapping rate,illumina,hiseq_era,unknown,poly_a,trueseq,bulk,unknown,unknown,,Germany,2022-01-01,Hatching,Embryo,Whole Organism,All anatomical structures
10322,ERR7179891,ERX6749448,ERS8138754,ERP132743,PRJEB48372,Transcriptomic profiling of clobetasol propionate induced immunosuppression in challenged zebrafish embryos,E-MTAB-11092,Transcriptome Analysis,The aim of this experiment was to develop an approach for the assessment of immunotoxicity in zebrafish embryos Danio rerio using transcriptomic profiling. Clobetasol propionate a highly potent synthetic corticosteroid which is known to have immunosuppressive effects on vertebrates including zebrafish was used for continuous exposure from fertilization until extraction. As immunotoxicity can only be comprehensively assessed when the effect is observed during an acute infection the embryos were injected with a mixture of different pathogen associated molecular patterns PAMPs consisting of Pam3CSK4 synthetic lipopeptide polyI:C synthetic viral dsRNA and flagellin isolated from Bacillus subtilis. Functional gene expression analysis from samples obtained by different kinds and combinations of treatments in relation to appropriate controls is used to identify pathways that are upregulated by artificial infection and suppressed by treatment with clobetasol propionate. In a modified version of the zebrafish embryo toxicity test OECD 236 20 fertilized eggs were exposed to a sub lethal concentration 250 nM of clobetasol propionate for xxx hours under semi static conditions. The test comprised of three treatments with clobetasol propionate and two negative control NC groups and was performed in triplicates. At 46 hpf the eggs were manually dechorinated. At 48 hpf the embryos were immobilized by anaesthetization using 200 mg/mL tricaine. Using microinjection 8 nL of the mixed PAMPs 0.33 µg/mL Pam3CSK4 0.33 µg/mL polyI:C and 0.033 µg/mL flagellin were injected directly into the embryos bloodstream. One of each exposure groups was injected with PAMPs while another one was injected with ultrapure water to control for effects introduced by the injection process. One control group remained completely untreated. post injection the embryos were further incubated for 3 hours before 10 embryos were randomly picked for each sample and pooled for RNA and protein extraction with NucleoSpin RNA/Protein kit Macherey Nagel. RNA quality was assessed with a 2100 Bioanalyzer system Agilent before coding RNA was purified PolyA selection with TruSeq RNA Library Prep Kit v2 and sequenced on an Illumina HiSeq 4000 System Illumina in 50 bp single read mode. Adapter sequences were removed with trimmomatic and sequences were aligned to the D.rerio reference genome GRCz11 with STAR. Counting of feature mapped reads was performed through featureCounts. Library gene count tables were then merged to a single count matrix as input for differential gene expression analysis with DESeq2.,ENA FIRST PUBLIC:2022 02 25|ENA LAST UPDATE:2022 02 25,,Protocols: For each sample 10 zebrafish larvae were randomly picked from the glass well and carefully transferred to a 1.5 ml low binding Eppendorf tube. Once individuals were collected for all samples the supernatant was removed and the tubes were snap frozen in liquid nitrogen. For detailed information about embryonic incubation conditions and husbandry of adult broodstock please refer to the “Growth protocol”. For detailed information about exposure duration and conditions please refer to the “Treatment protocol”. Adult animal husbandry: Wild type zebrafish Danio rerio Strain AB broodstocks were maintained under flow through conditions in 150 L tanks at 26 +/ 2°C on a 12:12 h light/dark cycle. They were fed daily with TetraMin® Tetra Werke Melle Germany main feed ad libitum and nauplii of Artemia salina. The day before test start glass spawning trays with artificial substrate green glass beads stringed on stainless steel wire were placed at the bottom of each tank. post mating and spawning in the morning hours eggs were rinsed with clear Cu reduced water and placed into glass dishes for pre picking. Fish embryo incubation: For each sample 20 fertilized eggs in the early blastula stage were placed in glass petri dishes diameter 6 cm filled with 8 mL of the testing solution or control medium. Embryos were incubated at 27 +/ 1°C on a 14:10 h light/dark cycle. At 24 hpf eggs were inspected while half of the test solution was renewed. Clobetasol propionate CAS: 25122 46 7 was purchased from Sigma Aldrich Taufkirchen Germany. For the test solution preparation first a 5 mM stock solution was prepared in Acetone 99.9% MS grade. This stock was diluted to 250 µM in acetone before 500 µL were added to a 500 mL DURAN glass bottle. To the bottle for the control solution only 500 µL of pure Acetone was added. post complete evaporation of the acetone the bottle was filled up with 500 mL copper reduced tap water. Complete dissolution of precipitated clobetasol propionate was achieved by sonication for 30 minutes Sonorex RK 100H Bandelin Germany. The test solutions were freshly prepared one day prior the start of the experiment and were constantly aerated at room temperature. The glass wells for the incubation of the embryos were pre saturated with the respective test solution overnight and renewed at the beginning of the experiment. Per sample about 40 50 fertilized fish eggs were pre picked in glass petri dishes 10 cm diameter filled with 30 ml of the testing solution. Using an optical binokular microscope 20 embryos of the same early blastula stage were transferred from the pre picking plate into the test glass wells filled with 8 ml of the test solution. Embryos were then incubated as described in the “growth protocol”. Standard flagellin from Bacillus subtilis ≈ 10% purity Pam3CSK4 ≥ 98 % purity and PolyI:C HMW high molecular weight were purchased from InvivoGen SAS Toulouse France. Stock solutions 1 mg/mL for polyI:C and Pam3CSK4; 0.1 mg/mL for flagellin were prepared in provided endotoxin free water aliquoted and stored according to the manufacturer's instructions until further use. At approximately 46 hpf the embryos were manually dechorinated and then further incubated. A mixture of the different PAMPs was prepared by mixing 10 µL of each component resulting in final concentrations of 0.33 mg/mL for polyI:C and Pam3CSK4 and 0.033 mg/mL for flagellin respectively. The device for microinjection was set up using FemtoTip II injection needles with a complementary universal capillary holder Eppendorf Hamburg Germany connected to an IM 300 microinjector Narishige London United Kingdom. Volume calibration was performed according to Sive et al. 2010 using a micrometer slide with 1/100 mm scale Bresser Rhede Germany. Briefly the needles were backfilled with 10 µL PAMP mix or ultra pure water. Injections for volume calibration were made into a drop of immersion oil purred on the micrometer slide the output pressure was constantly kept at 10 psi while the time of injection was adjusted until an injected drop had a diameter of approximately 250 µm. Assuming that the drop forms a perfect sphere this corresponds to a volume of about 8 nL. The procedure described below was performed for each of the five conditions in turn treating the three replicates simultaneously. In order to anaesthetize the embryos tricaine Sigma Aldrich Taufkirchen Germany stock solution 4 mg/mL in 21 mM Tris pH 9 was added to the petri dishes to a final concentration of 200 µg/mL. The immobilized embryos were transferred onto a 1 % agarose plate which was divided into segments one for each replicate. Excess liquid was removed so that the embryos were kept in place by surface tension. Desiccation was prevented by adding liquid if needed. To ensure that the injections could be performed as quickly as possible the embryos were arranged in a row in a way that their dorsal sides pointed to the same direction. The puncture was made on the dorsal side of the yolk sac and the needle was advanced through the yolk until it reached the yolk sac circulation valley on the ventral side of the embryo where the injection was made directly into the bloodstream. post all replicates of a certain condition were processed the embryos were rinsed back into the corresponding dish containing renewed test solution. Total RNA was extracted using the NucleoSpin RNA/Protein kit from Macherey Nagel 740933.250 The pooled and snap frozen larvae were suspended in RP1 buffer and were transferred to a screw cap tubes filled with 0.3 g of Lysing Matrix D MP Biomedicals 6913050 ceramic beads. Tissue homogenization was performed at 5 m/s for 45 s with FastPrep 24© MP Biomedicals 6004500 at room temperature. From here on RNA was extracted according to the manufacturer's protocol. RNA concentration >100 ng/µl was measured using a Nanodrop 2000 spectrophotometer Thermo Scientific. The sample's overall RNA quality and corresponding RIN value was fluorometrically determined with a 2100 Bioanalyzer© Instrument system Agilent G2939BA using the Agilent RNA 6000 Nano Kit© Agilent 5067 1511 according to the manufacturer's protocol. Only samples with a RIN > 8 were used for downstream analysis. Samples were stored at 80°C until they were send to the sequencing facility on dry ice. Sequencing libraries were prepared for each sample from 100 ng/µl total RNA at the sequencing facility “NGS Services for Integrative Genomics” at the University of Göttingen in Germany. According to their standard workflow cDNA libraries were prepared from protein coding mRNA that were purified through PolyA selection using the TruSeq RNA Library Prep Kit v2 Illumina. Libraries were validated using a Fragment Analyzer system Agilent Santa Clara USA before sequencing.,R1273,SAMEA10485996,"Fraunhofer Attract Eco'n'OMICs, Fraunhofer Institute for Molecular Biology and Applied Ecology, Schmallenberg, Germany",ENA first public:2022 01 01|ENA last update:2022 01 01|External Id:SAMEA10485996|INSDC center alias:Fraunhofer Attract Eco'n'OMICs Fraunhofer Institute for Molecular Biology and Applied Ecology Schmallenberg Germany|INSDC center name:Fraunhofer Attract Eco'n'OMICs Fraunhofer Institute for Molecular Biology and Applied Ecology Schmallenberg Germany|INSDC first public:2022 01 01T00:13:13Z|INSDC last update:2022 01 01T00:13:13Z|INSDC status:public|Submitter Id:E MTAB 11092:R1273|age:51|broker name:ArrayExpress|common name:zebrafish|developmental stage:hatching long pec|genotype:wild type genotype|organism part:whole organism|replicate:T12|sample name:E MTAB 11092:R1273|sex:mixed|stimulus:water|strain:AB,,,,,,,,,Illumina HiSeq 4000 sequencing; Transcriptomic profiling of clobetasol propionate induced immunosuppression in challenged zebrafish embryos,E MTAB 11092:R1273 s,R1273 s,Transcriptomic profiling of clobetasol propionate induced immunosuppression in challenged zebrafish embryos,For each sample 10 zebrafish larvae were randomly picked from the glass well and carefully transferred to a 1.5 ml low binding Eppendorf tube. Once individuals were collected for all samples the supernatant was removed and the tubes were snap frozen in liquid nitrogen. For detailed information about embryonic incubation conditions and husbandry of adult broodstock please refer to the Growth protocol. For detailed information about exposure duration and conditions please refer to the Treatment protocol. Adult animal husbandry: Wild type zebrafish Danio rerio Strain AB broodstocks were maintained under flow through conditions in 150 L tanks at 26 +/ 2C on a 12:12 h light/dark cycle. They were fed daily with TetraMin Tetra Werke Melle Germany main feed ad libitum and nauplii of Artemia salina. The day before test start glass spawning trays with artificial substrate green glass beads stringed on stainless steel wire were placed at the bottom of each tank. post mating and spawning in the morning hours eggs were rinsed with clear Cu reduced water and placed into glass dishes for pre picking. Fish embryo incubation: For each sample 20 fertilized eggs in the early blastula stage were placed in glass petri dishes diameter 6 cm filled with 8 mL of the testing solution or control medium. Embryos were incubated at 27 +/ 1C on a 14:10 h light/dark cycle. At 24 hpf eggs were inspected while half of the test solution was renewed. Clobetasol propionate CAS: 25122 46 7 was purchased from Sigma Aldrich Taufkirchen Germany. For the test solution preparation first a 5 mM stock solution was prepared in Acetone 99.9% MS grade. This stock was diluted to 250 M in acetone before 500 L were added to a 500 mL DURAN glass bottle. To the bottle for the control solution only 500 L of pure Acetone was added. post complete evaporation of the acetone the bottle was filled up with 500 mL copper reduced tap water. Complete dissolution of precipitated clobetasol propionate was achieved by sonication for 30 minutes Sonorex RK 100H Bandelin Germany. The test solutions were freshly prepared one day prior the start of the experiment and were constantly aerated at room temperature. The glass wells for the incubation of the embryos were pre saturated with the respective test solution overnight and renewed at the beginning of the experiment. Per sample about 40 50 fertilized fish eggs were pre picked in glass petri dishes 10 cm diameter filled with 30 ml of the testing solution. Using an optical binokular microscope 20 embryos of the same early blastula stage were transferred from the pre picking plate into the test glass wells filled with 8 ml of the test solution. Embryos were then incubated as described in the growth protocol. Standard flagellin from Bacillus subtilis 10% purity Pam3CSK4 98 % purity and PolyI:C HMW high molecular weight were purchased from InvivoGen SAS Toulouse France. Stock solutions 1 mg/mL for polyI:C and Pam3CSK4; 0.1 mg/mL for flagellin were prepared in provided endotoxin free water aliquoted and stored according to the manufacturers instructions until further use. At approximately 46 hpf the embryos were manually dechorinated and then further incubated. A mixture of the different PAMPs was prepared by mixing 10 L of each component resulting in final concentrations of 0.33 mg/mL for polyI:C and Pam3CSK4 and 0.033 mg/mL for flagellin respectively. The device for microinjection was set up using FemtoTip II injection needles with a complementary universal capillary holder Eppendorf Hamburg Germany connected to an IM 300 microinjector Narishige London United Kingdom. Volume calibration was performed according to Sive et al. 2010 using a micrometer slide with 1/100 mm scale Bresser Rhede Germany. Briefly the needles were backfilled with 10 L PAMP mix or ultra pure water. Injections for volume calibration were made into a drop of immersion oil purred on the micrometer slide the output pressure was constantly kept at 10 psi while the time of injection was adjusted until an injected drop had a diameter of approximately 250 m. Assuming that the drop forms a perfect sphere this corresponds to a volume of about 8 nL. The procedure described below was performed for each of the five conditions in turn treating the three replicates simultaneously. In order to anaesthetize the embryos tricaine Sigma Aldrich Taufkirchen Germany stock solution 4 mg/mL in 21 mM Tris pH 9 was added to the petri dishes to a final concentration of 200 g/mL. The immobilized embryos were transferred onto a 1 % agarose plate which was divided into segments one for each replicate. Excess liquid was removed so that the embryos were kept in place by surface tension. Desiccation was prevented by adding liquid if needed. To ensure that the injections could be performed as quickly as possible the embryos were arranged in a row in a way that their dorsal sides pointed to the same direction. The puncture was made on the dorsal side of the yolk sac and the needle was advanced through the yolk until it reached the yolk sac circulation valley on the ventral side of the embryo where the injection was made directly into the bloodstream. post all replicates of a certain condition were processed the embryos were rinsed back into the corresponding dish containing renewed test solution. Total RNA was extracted using the NucleoSpin RNA/Protein kit from Macherey Nagel 740933.250 The pooled and snap frozen larvae were suspended in RP1 buffer and were transferred to a screw cap tubes filled with 0.3 g of Lysing Matrix D MP Biomedicals 6913050 ceramic beads. Tissue homogenization was performed at 5 m/s for 45 s with FastPrep 24 MP Biomedicals 6004500 at room temperature. From here on RNA was extracted according to the manufacturers protocol. RNA concentration >100 ng/l was measured using a Nanodrop 2000 spectrophotometer Thermo Scientific. The samples overall RNA quality and corresponding RIN value was fluorometrically determined with a 2100 Bioanalyzer Instrument system Agilent G2939BA using the Agilent RNA 6000 Nano Kit Agilent 5067 1511 according to the manufacturers protocol. Only samples with a RIN > 8 were used for downstream analysis. Samples were stored at 80C until they were send to the sequencing facility on dry ice. Sequencing libraries were prepared for each sample from 100 ng/l total RNA at the sequencing facility NGS Services for Integrative Genomics at the University of Gttingen in Germany. According to their standard workflow cDNA libraries were prepared from protein coding mRNA that were purified through PolyA selection using the TruSeq RNA Library Prep Kit v2 Illumina. Libraries were validated using a Fragment Analyzer system Agilent Santa Clara USA before sequencing.,Experimental Factor: compound:n1|Experimental Factor: stimulus:water,RNA-Seq,TRANSCRIPTOMIC,PolyA,SINGLE,ILLUMINA,Illumina HiSeq 4000,,ERP132743,Illumina HiSeq 4000 sequencing; Transcriptomic profiling of clobetasol propionate induced immunosuppression in challenged zebrafish embryos,ENA FIRST PUBLIC:2022 02 25|ENA LAST UPDATE:2022 02 25|loader:fastq load.py,p946sR1273_sr.fastq.gz,fastq,978769211.0,19384726.0,E MTAB 11092:R1273,0:50.49,A:254759436;C:235060632;G:224944252;T:262789193;N:1215698,50,,,,254759436,235060632,224944252,262789193,1215698,ERX6749448,ERS8138754,ERA6860152,Fraunhofer Attract Eco,Fraunhofer Attract Eco,1,0.93731,,0.114,,0.68812,,0.46237,,51,,B,,usable mapping rate,illumina,hiseq_era,unknown,poly_a,trueseq,bulk,unknown,unknown,,Germany,2022-01-01,Hatching,Embryo,Whole Organism,All anatomical structures
10323,ERR7179890,ERX6749447,ERS8138753,ERP132743,PRJEB48372,Transcriptomic profiling of clobetasol propionate induced immunosuppression in challenged zebrafish embryos,E-MTAB-11092,Transcriptome Analysis,The aim of this experiment was to develop an approach for the assessment of immunotoxicity in zebrafish embryos Danio rerio using transcriptomic profiling. Clobetasol propionate a highly potent synthetic corticosteroid which is known to have immunosuppressive effects on vertebrates including zebrafish was used for continuous exposure from fertilization until extraction. As immunotoxicity can only be comprehensively assessed when the effect is observed during an acute infection the embryos were injected with a mixture of different pathogen associated molecular patterns PAMPs consisting of Pam3CSK4 synthetic lipopeptide polyI:C synthetic viral dsRNA and flagellin isolated from Bacillus subtilis. Functional gene expression analysis from samples obtained by different kinds and combinations of treatments in relation to appropriate controls is used to identify pathways that are upregulated by artificial infection and suppressed by treatment with clobetasol propionate. In a modified version of the zebrafish embryo toxicity test OECD 236 20 fertilized eggs were exposed to a sub lethal concentration 250 nM of clobetasol propionate for xxx hours under semi static conditions. The test comprised of three treatments with clobetasol propionate and two negative control NC groups and was performed in triplicates. At 46 hpf the eggs were manually dechorinated. At 48 hpf the embryos were immobilized by anaesthetization using 200 mg/mL tricaine. Using microinjection 8 nL of the mixed PAMPs 0.33 µg/mL Pam3CSK4 0.33 µg/mL polyI:C and 0.033 µg/mL flagellin were injected directly into the embryos bloodstream. One of each exposure groups was injected with PAMPs while another one was injected with ultrapure water to control for effects introduced by the injection process. One control group remained completely untreated. post injection the embryos were further incubated for 3 hours before 10 embryos were randomly picked for each sample and pooled for RNA and protein extraction with NucleoSpin RNA/Protein kit Macherey Nagel. RNA quality was assessed with a 2100 Bioanalyzer system Agilent before coding RNA was purified PolyA selection with TruSeq RNA Library Prep Kit v2 and sequenced on an Illumina HiSeq 4000 System Illumina in 50 bp single read mode. Adapter sequences were removed with trimmomatic and sequences were aligned to the D.rerio reference genome GRCz11 with STAR. Counting of feature mapped reads was performed through featureCounts. Library gene count tables were then merged to a single count matrix as input for differential gene expression analysis with DESeq2.,ENA FIRST PUBLIC:2022 02 25|ENA LAST UPDATE:2022 02 25,,Protocols: For each sample 10 zebrafish larvae were randomly picked from the glass well and carefully transferred to a 1.5 ml low binding Eppendorf tube. Once individuals were collected for all samples the supernatant was removed and the tubes were snap frozen in liquid nitrogen. For detailed information about embryonic incubation conditions and husbandry of adult broodstock please refer to the “Growth protocol”. For detailed information about exposure duration and conditions please refer to the “Treatment protocol”. Adult animal husbandry: Wild type zebrafish Danio rerio Strain AB broodstocks were maintained under flow through conditions in 150 L tanks at 26 +/ 2°C on a 12:12 h light/dark cycle. They were fed daily with TetraMin® Tetra Werke Melle Germany main feed ad libitum and nauplii of Artemia salina. The day before test start glass spawning trays with artificial substrate green glass beads stringed on stainless steel wire were placed at the bottom of each tank. post mating and spawning in the morning hours eggs were rinsed with clear Cu reduced water and placed into glass dishes for pre picking. Fish embryo incubation: For each sample 20 fertilized eggs in the early blastula stage were placed in glass petri dishes diameter 6 cm filled with 8 mL of the testing solution or control medium. Embryos were incubated at 27 +/ 1°C on a 14:10 h light/dark cycle. At 24 hpf eggs were inspected while half of the test solution was renewed. Clobetasol propionate CAS: 25122 46 7 was purchased from Sigma Aldrich Taufkirchen Germany. For the test solution preparation first a 5 mM stock solution was prepared in Acetone 99.9% MS grade. This stock was diluted to 250 µM in acetone before 500 µL were added to a 500 mL DURAN glass bottle. To the bottle for the control solution only 500 µL of pure Acetone was added. post complete evaporation of the acetone the bottle was filled up with 500 mL copper reduced tap water. Complete dissolution of precipitated clobetasol propionate was achieved by sonication for 30 minutes Sonorex RK 100H Bandelin Germany. The test solutions were freshly prepared one day prior the start of the experiment and were constantly aerated at room temperature. The glass wells for the incubation of the embryos were pre saturated with the respective test solution overnight and renewed at the beginning of the experiment. Per sample about 40 50 fertilized fish eggs were pre picked in glass petri dishes 10 cm diameter filled with 30 ml of the testing solution. Using an optical binokular microscope 20 embryos of the same early blastula stage were transferred from the pre picking plate into the test glass wells filled with 8 ml of the test solution. Embryos were then incubated as described in the “growth protocol”. Standard flagellin from Bacillus subtilis ≈ 10% purity Pam3CSK4 ≥ 98 % purity and PolyI:C HMW high molecular weight were purchased from InvivoGen SAS Toulouse France. Stock solutions 1 mg/mL for polyI:C and Pam3CSK4; 0.1 mg/mL for flagellin were prepared in provided endotoxin free water aliquoted and stored according to the manufacturer's instructions until further use. At approximately 46 hpf the embryos were manually dechorinated and then further incubated. A mixture of the different PAMPs was prepared by mixing 10 µL of each component resulting in final concentrations of 0.33 mg/mL for polyI:C and Pam3CSK4 and 0.033 mg/mL for flagellin respectively. The device for microinjection was set up using FemtoTip II injection needles with a complementary universal capillary holder Eppendorf Hamburg Germany connected to an IM 300 microinjector Narishige London United Kingdom. Volume calibration was performed according to Sive et al. 2010 using a micrometer slide with 1/100 mm scale Bresser Rhede Germany. Briefly the needles were backfilled with 10 µL PAMP mix or ultra pure water. Injections for volume calibration were made into a drop of immersion oil purred on the micrometer slide the output pressure was constantly kept at 10 psi while the time of injection was adjusted until an injected drop had a diameter of approximately 250 µm. Assuming that the drop forms a perfect sphere this corresponds to a volume of about 8 nL. The procedure described below was performed for each of the five conditions in turn treating the three replicates simultaneously. In order to anaesthetize the embryos tricaine Sigma Aldrich Taufkirchen Germany stock solution 4 mg/mL in 21 mM Tris pH 9 was added to the petri dishes to a final concentration of 200 µg/mL. The immobilized embryos were transferred onto a 1 % agarose plate which was divided into segments one for each replicate. Excess liquid was removed so that the embryos were kept in place by surface tension. Desiccation was prevented by adding liquid if needed. To ensure that the injections could be performed as quickly as possible the embryos were arranged in a row in a way that their dorsal sides pointed to the same direction. The puncture was made on the dorsal side of the yolk sac and the needle was advanced through the yolk until it reached the yolk sac circulation valley on the ventral side of the embryo where the injection was made directly into the bloodstream. post all replicates of a certain condition were processed the embryos were rinsed back into the corresponding dish containing renewed test solution. Total RNA was extracted using the NucleoSpin RNA/Protein kit from Macherey Nagel 740933.250 The pooled and snap frozen larvae were suspended in RP1 buffer and were transferred to a screw cap tubes filled with 0.3 g of Lysing Matrix D MP Biomedicals 6913050 ceramic beads. Tissue homogenization was performed at 5 m/s for 45 s with FastPrep 24© MP Biomedicals 6004500 at room temperature. From here on RNA was extracted according to the manufacturer's protocol. RNA concentration >100 ng/µl was measured using a Nanodrop 2000 spectrophotometer Thermo Scientific. The sample's overall RNA quality and corresponding RIN value was fluorometrically determined with a 2100 Bioanalyzer© Instrument system Agilent G2939BA using the Agilent RNA 6000 Nano Kit© Agilent 5067 1511 according to the manufacturer's protocol. Only samples with a RIN > 8 were used for downstream analysis. Samples were stored at 80°C until they were send to the sequencing facility on dry ice. Sequencing libraries were prepared for each sample from 100 ng/µl total RNA at the sequencing facility “NGS Services for Integrative Genomics” at the University of Göttingen in Germany. According to their standard workflow cDNA libraries were prepared from protein coding mRNA that were purified through PolyA selection using the TruSeq RNA Library Prep Kit v2 Illumina. Libraries were validated using a Fragment Analyzer system Agilent Santa Clara USA before sequencing.,R1270,SAMEA10485994,"Fraunhofer Attract Eco'n'OMICs, Fraunhofer Institute for Molecular Biology and Applied Ecology, Schmallenberg, Germany",ENA first public:2022 01 01|ENA last update:2022 01 01|External Id:SAMEA10485994|INSDC center alias:Fraunhofer Attract Eco'n'OMICs Fraunhofer Institute for Molecular Biology and Applied Ecology Schmallenberg Germany|INSDC center name:Fraunhofer Attract Eco'n'OMICs Fraunhofer Institute for Molecular Biology and Applied Ecology Schmallenberg Germany|INSDC first public:2022 01 01T00:13:13Z|INSDC last update:2022 01 01T00:13:13Z|INSDC status:public|Submitter Id:E MTAB 11092:R1270|age:51|broker name:ArrayExpress|common name:zebrafish|developmental stage:hatching long pec|genotype:wild type genotype|organism part:whole organism|replicate:T12|sample name:E MTAB 11092:R1270|sex:mixed|stimulus:control|strain:AB,,,,,,,,,Illumina HiSeq 4000 sequencing; Transcriptomic profiling of clobetasol propionate induced immunosuppression in challenged zebrafish embryos,E MTAB 11092:R1270 s,R1270 s,Transcriptomic profiling of clobetasol propionate induced immunosuppression in challenged zebrafish embryos,For each sample 10 zebrafish larvae were randomly picked from the glass well and carefully transferred to a 1.5 ml low binding Eppendorf tube. Once individuals were collected for all samples the supernatant was removed and the tubes were snap frozen in liquid nitrogen. For detailed information about embryonic incubation conditions and husbandry of adult broodstock please refer to the Growth protocol. For detailed information about exposure duration and conditions please refer to the Treatment protocol. Adult animal husbandry: Wild type zebrafish Danio rerio Strain AB broodstocks were maintained under flow through conditions in 150 L tanks at 26 +/ 2C on a 12:12 h light/dark cycle. They were fed daily with TetraMin Tetra Werke Melle Germany main feed ad libitum and nauplii of Artemia salina. The day before test start glass spawning trays with artificial substrate green glass beads stringed on stainless steel wire were placed at the bottom of each tank. post mating and spawning in the morning hours eggs were rinsed with clear Cu reduced water and placed into glass dishes for pre picking. Fish embryo incubation: For each sample 20 fertilized eggs in the early blastula stage were placed in glass petri dishes diameter 6 cm filled with 8 mL of the testing solution or control medium. Embryos were incubated at 27 +/ 1C on a 14:10 h light/dark cycle. At 24 hpf eggs were inspected while half of the test solution was renewed. Clobetasol propionate CAS: 25122 46 7 was purchased from Sigma Aldrich Taufkirchen Germany. For the test solution preparation first a 5 mM stock solution was prepared in Acetone 99.9% MS grade. This stock was diluted to 250 M in acetone before 500 L were added to a 500 mL DURAN glass bottle. To the bottle for the control solution only 500 L of pure Acetone was added. post complete evaporation of the acetone the bottle was filled up with 500 mL copper reduced tap water. Complete dissolution of precipitated clobetasol propionate was achieved by sonication for 30 minutes Sonorex RK 100H Bandelin Germany. The test solutions were freshly prepared one day prior the start of the experiment and were constantly aerated at room temperature. The glass wells for the incubation of the embryos were pre saturated with the respective test solution overnight and renewed at the beginning of the experiment. Per sample about 40 50 fertilized fish eggs were pre picked in glass petri dishes 10 cm diameter filled with 30 ml of the testing solution. Using an optical binokular microscope 20 embryos of the same early blastula stage were transferred from the pre picking plate into the test glass wells filled with 8 ml of the test solution. Embryos were then incubated as described in the growth protocol. Standard flagellin from Bacillus subtilis 10% purity Pam3CSK4 98 % purity and PolyI:C HMW high molecular weight were purchased from InvivoGen SAS Toulouse France. Stock solutions 1 mg/mL for polyI:C and Pam3CSK4; 0.1 mg/mL for flagellin were prepared in provided endotoxin free water aliquoted and stored according to the manufacturers instructions until further use. At approximately 46 hpf the embryos were manually dechorinated and then further incubated. A mixture of the different PAMPs was prepared by mixing 10 L of each component resulting in final concentrations of 0.33 mg/mL for polyI:C and Pam3CSK4 and 0.033 mg/mL for flagellin respectively. The device for microinjection was set up using FemtoTip II injection needles with a complementary universal capillary holder Eppendorf Hamburg Germany connected to an IM 300 microinjector Narishige London United Kingdom. Volume calibration was performed according to Sive et al. 2010 using a micrometer slide with 1/100 mm scale Bresser Rhede Germany. Briefly the needles were backfilled with 10 L PAMP mix or ultra pure water. Injections for volume calibration were made into a drop of immersion oil purred on the micrometer slide the output pressure was constantly kept at 10 psi while the time of injection was adjusted until an injected drop had a diameter of approximately 250 m. Assuming that the drop forms a perfect sphere this corresponds to a volume of about 8 nL. The procedure described below was performed for each of the five conditions in turn treating the three replicates simultaneously. In order to anaesthetize the embryos tricaine Sigma Aldrich Taufkirchen Germany stock solution 4 mg/mL in 21 mM Tris pH 9 was added to the petri dishes to a final concentration of 200 g/mL. The immobilized embryos were transferred onto a 1 % agarose plate which was divided into segments one for each replicate. Excess liquid was removed so that the embryos were kept in place by surface tension. Desiccation was prevented by adding liquid if needed. To ensure that the injections could be performed as quickly as possible the embryos were arranged in a row in a way that their dorsal sides pointed to the same direction. The puncture was made on the dorsal side of the yolk sac and the needle was advanced through the yolk until it reached the yolk sac circulation valley on the ventral side of the embryo where the injection was made directly into the bloodstream. post all replicates of a certain condition were processed the embryos were rinsed back into the corresponding dish containing renewed test solution. Total RNA was extracted using the NucleoSpin RNA/Protein kit from Macherey Nagel 740933.250 The pooled and snap frozen larvae were suspended in RP1 buffer and were transferred to a screw cap tubes filled with 0.3 g of Lysing Matrix D MP Biomedicals 6913050 ceramic beads. Tissue homogenization was performed at 5 m/s for 45 s with FastPrep 24 MP Biomedicals 6004500 at room temperature. From here on RNA was extracted according to the manufacturers protocol. RNA concentration >100 ng/l was measured using a Nanodrop 2000 spectrophotometer Thermo Scientific. The samples overall RNA quality and corresponding RIN value was fluorometrically determined with a 2100 Bioanalyzer Instrument system Agilent G2939BA using the Agilent RNA 6000 Nano Kit Agilent 5067 1511 according to the manufacturers protocol. Only samples with a RIN > 8 were used for downstream analysis. Samples were stored at 80C until they were send to the sequencing facility on dry ice. Sequencing libraries were prepared for each sample from 100 ng/l total RNA at the sequencing facility NGS Services for Integrative Genomics at the University of Gttingen in Germany. According to their standard workflow cDNA libraries were prepared from protein coding mRNA that were purified through PolyA selection using the TruSeq RNA Library Prep Kit v2 Illumina. Libraries were validated using a Fragment Analyzer system Agilent Santa Clara USA before sequencing.,Experimental Factor: compound:n1|Experimental Factor: stimulus:n1,RNA-Seq,TRANSCRIPTOMIC,PolyA,SINGLE,ILLUMINA,Illumina HiSeq 4000,,ERP132743,Illumina HiSeq 4000 sequencing; Transcriptomic profiling of clobetasol propionate induced immunosuppression in challenged zebrafish embryos,ENA FIRST PUBLIC:2022 02 25|ENA LAST UPDATE:2022 02 25|loader:fastq load.py,p946sR1270_sr.fastq.gz,fastq,1079683414.0,21379121.0,E MTAB 11092:R1270,0:50.50,A:282457582;C:258094012;G:247325987;T:291030099;N:775734,50,,,,282457582,258094012,247325987,291030099,775734,ERX6749447,ERS8138753,ERA6860152,Fraunhofer Attract Eco,Fraunhofer Attract Eco,1,0.93758,,0.10619,,0.68363,,0.47429,,51,,B,,usable mapping rate,illumina,hiseq_era,unknown,poly_a,trueseq,bulk,unknown,unknown,,Germany,2022-01-01,Hatching,Embryo,Whole Organism,All anatomical structures
15009,ERR12306826,ERX11683773,ERS17043893,ERP155237,PRJEB70303,10X genomics libraries for scRNA seq of zebrafish embryos 2dpf treated with uncoated and oligolysine PEG copolymer coated DNA origami wireframe nanosheets,E-MTAB-13554,Transcriptome Analysis,To study the biodistribution of DNA origami in zebrafish and to determine differences in biodistribution based on the coating of the nanostructure using oligolysine PEG copolymers zebrafish embryos 2dpf were injected with coated or uncoated DNA origami wireframe nanosheets without xxx targeting molecule but containing a detection fluorophores. post treatment the embryos were disassociated into a single cell suspension and sorted through FACS for embryos containing nanostructures fluorophores. 10X sc libraries were generated from the sorted embryos and sequenced to establish cell type predictions for cells with coated and uncoated structures respectively.,ENA FIRST PUBLIC:2024 09 30|ENA LAST UPDATE:2024 09 30,,Protocols: Embryos 48 hpf were disassociated into a single cell suspension. Four hours post injections around 200 Tgfli1:EGFP embryos were euthanized in 0.02% tricaine. The tissue was dissociated with harsh pipetting in 0.25% trypsin EDTA GIBCO and 100mg/mL collagenase Sigma Aldrich for 3 min at 30oC followed by resuspension in DMEM 10% FBS. The cells were then pelleted at 700xg for 5 min washed once in 1X PBS filtered through a 40 μm cell strainer MERCK and stained with 1mM DAPI solution Abcam. TS+ cells were FACS sorted on a FACSARIA III with BD FACSDIVA software v. 9.0.1 BD Biosciences. Zebrafish D. rerio were maintained and bred in compliance with Swedish legislation on animal welfare regulations approved by Stockholms djurförsöksetiska nämnd. Each zebrafish embryo Tgfli1:EGFP was microinjected with 4 nl of the sample. The samples for injections were prepared at a final concentration of 100 nM in 1x PBS effective dose estimated to around 4 6 nM. Embryos at 48 hpf were anesthetized using 0.1% tricaine MS222 transferred onto an agarose surface placed on their ventral side and injected into the common cardinal vein Duct of Cuvier. scRNAseq libraries were generated from the sorted cells using 10X Genomics Chromium Single Cell 3ʹ Reagent Kits v3.1 per manufacturer's instructions targeting 8000 cells. The cDNA libraries were amplified using 10X Genomics Chromium Single Cell 3ʹ Reagent Kits v3.1 with 14 PCR cycles.,Coated,SAMEA114641726,KIS,ENA first public:2024 09 30|External Id:SAMEA114641726|INSDC center name:KIS|INSDC last update:2023 11 20T16:06:50Z|INSDC status:public|Submitter Id:E MTAB 13554:Coated|age:2|broker name:ArrayExpress|collection date:not collected|common name:zebrafish|developmental stage:embryo stage|geographic location country and/or sea:not collected|isolate:not applicable|organism part:whole organism|sample name:E MTAB 13554:Coated|scientific name:Danio rerio|sex:not available|strain:transgenic lines Tgfli1:EGFPy1,,,,,,,,,10X genomics libraries for scRNA seq of zebrafish embryos 2dpf treated with uncoated and oligolysine PEG copolymer coated DNA origami wireframe nanosheets,E MTAB 13554:Coated p,Coated p,10X genomics libraries for scRNA seq of zebrafish embryos 2dpf treated with uncoated and oligolysine PEG copolymer coated DNA origami wireframe nanosheets,Embryos 48 hpf were disassociated into a single cell suspension. Four hours post injections around 200 Tgfli1:EGFP embryos were euthanized in 0.02% tricaine. The tissue was dissociated with harsh pipetting in 0.25% trypsin EDTA GIBCO and 100mg/mL collagenase Sigma Aldrich for 3 min at 30oC followed by resuspension in DMEM 10% FBS. The cells were then pelleted at 700xg for 5 min washed once in 1X PBS filtered through a 40 μm cell strainer MERCK and stained with 1mM DAPI solution Abcam. TS+ cells were FACS sorted on a FACSARIA III with BD FACSDIVA software v. 9.0.1 BD Biosciences. Zebrafish D. rerio were maintained and bred in compliance with Swedish legislation on animal welfare regulations approved by Stockholms djurförsöksetiska nämnd. Each zebrafish embryo Tgfli1:EGFP was microinjected with 4 nl of the sample. The samples for injections were prepared at a final concentration of 100 nM in 1x PBS effective dose estimated to around 4 6 nM. Embryos at 48 hpf were anesthetized using 0.1% tricaine MS222 transferred onto an agarose surface placed on their ventral side and injected into the common cardinal vein Duct of Cuvier. scRNAseq libraries were generated from the sorted cells using 10X Genomics Chromium Single Cell 3ʹ Reagent Kits v3.1 per manufacturer's instructions targeting 8000 cells. The cDNA libraries were amplified using 10X Genomics Chromium Single Cell 3ʹ Reagent Kits v3.1 with 14 PCR cycles.,,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,PolyA,PAIRED,ILLUMINA,NextSeq 550,,ERP155237,NextSeq 550 paired end sequencing; 10X genomics libraries for scRNA seq of zebrafish embryos 2dpf treated with uncoated and oligolysine PEG copolymer coated DNA origami wireframe nanosheets,ENA FIRST PUBLIC:2024 09 30|ENA LAST UPDATE:2024 09 30,230324-Coated_S1_L003_I1_001.fastq.gz 230324-Coated_S1_L003_I2_001.fastq.gz 230324-Coated_S1_L003_R1_001.fastq.gz 230324-Coated_S1_L003_R2_001.fastq.gz,fastq fastq fastq fastq,10050750400.0,62817190.0,E MTAB 13554:230324 Coated S1 L003,0:10 1:10 2:30 3:110,A:1894211982;C:1556769050;G:1595134885;T:1863244619;N:530364,10,10,30,110,1894211982,1556769050,1595134885,1863244619,530364,ERX11683773,ERS17043893,ERA27417303,European Bioinformatics Institute|European Nucleotide Archive,European Bioinformatics Institute|European Nucleotide Archive,,,,,,,,,,,,B,,usable mapping rate,illumina,nextseq,unknown,poly_a,unknown,sc,single_cell_droplet,10x,,United Kingdom,2023-11-20,Hatching,Embryo,Whole Organism,All anatomical structures
15010,ERR12306829,ERX11683773,ERS17043893,ERP155237,PRJEB70303,10X genomics libraries for scRNA seq of zebrafish embryos 2dpf treated with uncoated and oligolysine PEG copolymer coated DNA origami wireframe nanosheets,E-MTAB-13554,Transcriptome Analysis,To study the biodistribution of DNA origami in zebrafish and to determine differences in biodistribution based on the coating of the nanostructure using oligolysine PEG copolymers zebrafish embryos 2dpf were injected with coated or uncoated DNA origami wireframe nanosheets without xxx targeting molecule but containing a detection fluorophores. post treatment the embryos were disassociated into a single cell suspension and sorted through FACS for embryos containing nanostructures fluorophores. 10X sc libraries were generated from the sorted embryos and sequenced to establish cell type predictions for cells with coated and uncoated structures respectively.,ENA FIRST PUBLIC:2024 09 30|ENA LAST UPDATE:2024 09 30,,Protocols: Embryos 48 hpf were disassociated into a single cell suspension. Four hours post injections around 200 Tgfli1:EGFP embryos were euthanized in 0.02% tricaine. The tissue was dissociated with harsh pipetting in 0.25% trypsin EDTA GIBCO and 100mg/mL collagenase Sigma Aldrich for 3 min at 30oC followed by resuspension in DMEM 10% FBS. The cells were then pelleted at 700xg for 5 min washed once in 1X PBS filtered through a 40 μm cell strainer MERCK and stained with 1mM DAPI solution Abcam. TS+ cells were FACS sorted on a FACSARIA III with BD FACSDIVA software v. 9.0.1 BD Biosciences. Zebrafish D. rerio were maintained and bred in compliance with Swedish legislation on animal welfare regulations approved by Stockholms djurförsöksetiska nämnd. Each zebrafish embryo Tgfli1:EGFP was microinjected with 4 nl of the sample. The samples for injections were prepared at a final concentration of 100 nM in 1x PBS effective dose estimated to around 4 6 nM. Embryos at 48 hpf were anesthetized using 0.1% tricaine MS222 transferred onto an agarose surface placed on their ventral side and injected into the common cardinal vein Duct of Cuvier. scRNAseq libraries were generated from the sorted cells using 10X Genomics Chromium Single Cell 3ʹ Reagent Kits v3.1 per manufacturer's instructions targeting 8000 cells. The cDNA libraries were amplified using 10X Genomics Chromium Single Cell 3ʹ Reagent Kits v3.1 with 14 PCR cycles.,Coated,SAMEA114641726,KIS,ENA first public:2024 09 30|External Id:SAMEA114641726|INSDC center name:KIS|INSDC last update:2023 11 20T16:06:50Z|INSDC status:public|Submitter Id:E MTAB 13554:Coated|age:2|broker name:ArrayExpress|collection date:not collected|common name:zebrafish|developmental stage:embryo stage|geographic location country and/or sea:not collected|isolate:not applicable|organism part:whole organism|sample name:E MTAB 13554:Coated|scientific name:Danio rerio|sex:not available|strain:transgenic lines Tgfli1:EGFPy1,,,,,,,,,10X genomics libraries for scRNA seq of zebrafish embryos 2dpf treated with uncoated and oligolysine PEG copolymer coated DNA origami wireframe nanosheets,E MTAB 13554:Coated p,Coated p,10X genomics libraries for scRNA seq of zebrafish embryos 2dpf treated with uncoated and oligolysine PEG copolymer coated DNA origami wireframe nanosheets,Embryos 48 hpf were disassociated into a single cell suspension. Four hours post injections around 200 Tgfli1:EGFP embryos were euthanized in 0.02% tricaine. The tissue was dissociated with harsh pipetting in 0.25% trypsin EDTA GIBCO and 100mg/mL collagenase Sigma Aldrich for 3 min at 30oC followed by resuspension in DMEM 10% FBS. The cells were then pelleted at 700xg for 5 min washed once in 1X PBS filtered through a 40 μm cell strainer MERCK and stained with 1mM DAPI solution Abcam. TS+ cells were FACS sorted on a FACSARIA III with BD FACSDIVA software v. 9.0.1 BD Biosciences. Zebrafish D. rerio were maintained and bred in compliance with Swedish legislation on animal welfare regulations approved by Stockholms djurförsöksetiska nämnd. Each zebrafish embryo Tgfli1:EGFP was microinjected with 4 nl of the sample. The samples for injections were prepared at a final concentration of 100 nM in 1x PBS effective dose estimated to around 4 6 nM. Embryos at 48 hpf were anesthetized using 0.1% tricaine MS222 transferred onto an agarose surface placed on their ventral side and injected into the common cardinal vein Duct of Cuvier. scRNAseq libraries were generated from the sorted cells using 10X Genomics Chromium Single Cell 3ʹ Reagent Kits v3.1 per manufacturer's instructions targeting 8000 cells. The cDNA libraries were amplified using 10X Genomics Chromium Single Cell 3ʹ Reagent Kits v3.1 with 14 PCR cycles.,,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,PolyA,PAIRED,ILLUMINA,NextSeq 550,,ERP155237,NextSeq 550 paired end sequencing; 10X genomics libraries for scRNA seq of zebrafish embryos 2dpf treated with uncoated and oligolysine PEG copolymer coated DNA origami wireframe nanosheets,ENA FIRST PUBLIC:2024 09 30|ENA LAST UPDATE:2024 09 30,230324-Coated_S1_L004_I1_001.fastq.gz 230324-Coated_S1_L004_I2_001.fastq.gz 230324-Coated_S1_L004_R1_001.fastq.gz 230324-Coated_S1_L004_R2_001.fastq.gz,fastq fastq fastq fastq,9780757760.0,61129736.0,E MTAB 13554:230324 Coated S1 L004,0:10 1:10 2:30 3:110,A:1844808667;C:1516149264;G:1549838165;T:1813091860;N:383004,10,10,30,110,1844808667,1516149264,1549838165,1813091860,383004,ERX11683773,ERS17043893,ERA27417303,European Bioinformatics Institute|European Nucleotide Archive,European Bioinformatics Institute|European Nucleotide Archive,,,,,,,,,,,,B,,usable mapping rate,illumina,nextseq,unknown,poly_a,unknown,sc,single_cell_droplet,10x,,United Kingdom,2023-11-20,Hatching,Embryo,Whole Organism,All anatomical structures
15011,ERR12306828,ERX11683773,ERS17043893,ERP155237,PRJEB70303,10X genomics libraries for scRNA seq of zebrafish embryos 2dpf treated with uncoated and oligolysine PEG copolymer coated DNA origami wireframe nanosheets,E-MTAB-13554,Transcriptome Analysis,To study the biodistribution of DNA origami in zebrafish and to determine differences in biodistribution based on the coating of the nanostructure using oligolysine PEG copolymers zebrafish embryos 2dpf were injected with coated or uncoated DNA origami wireframe nanosheets without xxx targeting molecule but containing a detection fluorophores. post treatment the embryos were disassociated into a single cell suspension and sorted through FACS for embryos containing nanostructures fluorophores. 10X sc libraries were generated from the sorted embryos and sequenced to establish cell type predictions for cells with coated and uncoated structures respectively.,ENA FIRST PUBLIC:2024 09 30|ENA LAST UPDATE:2024 09 30,,Protocols: Embryos 48 hpf were disassociated into a single cell suspension. Four hours post injections around 200 Tgfli1:EGFP embryos were euthanized in 0.02% tricaine. The tissue was dissociated with harsh pipetting in 0.25% trypsin EDTA GIBCO and 100mg/mL collagenase Sigma Aldrich for 3 min at 30oC followed by resuspension in DMEM 10% FBS. The cells were then pelleted at 700xg for 5 min washed once in 1X PBS filtered through a 40 μm cell strainer MERCK and stained with 1mM DAPI solution Abcam. TS+ cells were FACS sorted on a FACSARIA III with BD FACSDIVA software v. 9.0.1 BD Biosciences. Zebrafish D. rerio were maintained and bred in compliance with Swedish legislation on animal welfare regulations approved by Stockholms djurförsöksetiska nämnd. Each zebrafish embryo Tgfli1:EGFP was microinjected with 4 nl of the sample. The samples for injections were prepared at a final concentration of 100 nM in 1x PBS effective dose estimated to around 4 6 nM. Embryos at 48 hpf were anesthetized using 0.1% tricaine MS222 transferred onto an agarose surface placed on their ventral side and injected into the common cardinal vein Duct of Cuvier. scRNAseq libraries were generated from the sorted cells using 10X Genomics Chromium Single Cell 3ʹ Reagent Kits v3.1 per manufacturer's instructions targeting 8000 cells. The cDNA libraries were amplified using 10X Genomics Chromium Single Cell 3ʹ Reagent Kits v3.1 with 14 PCR cycles.,Coated,SAMEA114641726,KIS,ENA first public:2024 09 30|External Id:SAMEA114641726|INSDC center name:KIS|INSDC last update:2023 11 20T16:06:50Z|INSDC status:public|Submitter Id:E MTAB 13554:Coated|age:2|broker name:ArrayExpress|collection date:not collected|common name:zebrafish|developmental stage:embryo stage|geographic location country and/or sea:not collected|isolate:not applicable|organism part:whole organism|sample name:E MTAB 13554:Coated|scientific name:Danio rerio|sex:not available|strain:transgenic lines Tgfli1:EGFPy1,,,,,,,,,10X genomics libraries for scRNA seq of zebrafish embryos 2dpf treated with uncoated and oligolysine PEG copolymer coated DNA origami wireframe nanosheets,E MTAB 13554:Coated p,Coated p,10X genomics libraries for scRNA seq of zebrafish embryos 2dpf treated with uncoated and oligolysine PEG copolymer coated DNA origami wireframe nanosheets,Embryos 48 hpf were disassociated into a single cell suspension. Four hours post injections around 200 Tgfli1:EGFP embryos were euthanized in 0.02% tricaine. The tissue was dissociated with harsh pipetting in 0.25% trypsin EDTA GIBCO and 100mg/mL collagenase Sigma Aldrich for 3 min at 30oC followed by resuspension in DMEM 10% FBS. The cells were then pelleted at 700xg for 5 min washed once in 1X PBS filtered through a 40 μm cell strainer MERCK and stained with 1mM DAPI solution Abcam. TS+ cells were FACS sorted on a FACSARIA III with BD FACSDIVA software v. 9.0.1 BD Biosciences. Zebrafish D. rerio were maintained and bred in compliance with Swedish legislation on animal welfare regulations approved by Stockholms djurförsöksetiska nämnd. Each zebrafish embryo Tgfli1:EGFP was microinjected with 4 nl of the sample. The samples for injections were prepared at a final concentration of 100 nM in 1x PBS effective dose estimated to around 4 6 nM. Embryos at 48 hpf were anesthetized using 0.1% tricaine MS222 transferred onto an agarose surface placed on their ventral side and injected into the common cardinal vein Duct of Cuvier. scRNAseq libraries were generated from the sorted cells using 10X Genomics Chromium Single Cell 3ʹ Reagent Kits v3.1 per manufacturer's instructions targeting 8000 cells. The cDNA libraries were amplified using 10X Genomics Chromium Single Cell 3ʹ Reagent Kits v3.1 with 14 PCR cycles.,,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,PolyA,PAIRED,ILLUMINA,NextSeq 550,,ERP155237,NextSeq 550 paired end sequencing; 10X genomics libraries for scRNA seq of zebrafish embryos 2dpf treated with uncoated and oligolysine PEG copolymer coated DNA origami wireframe nanosheets,ENA FIRST PUBLIC:2024 09 30|ENA LAST UPDATE:2024 09 30,230324-Coated_S1_L002_I1_001.fastq.gz 230324-Coated_S1_L002_I2_001.fastq.gz 230324-Coated_S1_L002_R1_001.fastq.gz 230324-Coated_S1_L002_R2_001.fastq.gz,fastq fastq fastq fastq,9639368480.0,60246053.0,E MTAB 13554:230324 Coated S1 L002,0:10 1:10 2:30 3:110,A:1817388578;C:1494319441;G:1527564319;T:1787447672;N:345820,10,10,30,110,1817388578,1494319441,1527564319,1787447672,345820,ERX11683773,ERS17043893,ERA27417303,European Bioinformatics Institute|European Nucleotide Archive,European Bioinformatics Institute|European Nucleotide Archive,,,,,,,,,,,,B,,usable mapping rate,illumina,nextseq,unknown,poly_a,unknown,sc,single_cell_droplet,10x,,United Kingdom,2023-11-20,Hatching,Embryo,Whole Organism,All anatomical structures
15012,ERR12306832,ERX11683773,ERS17043893,ERP155237,PRJEB70303,10X genomics libraries for scRNA seq of zebrafish embryos 2dpf treated with uncoated and oligolysine PEG copolymer coated DNA origami wireframe nanosheets,E-MTAB-13554,Transcriptome Analysis,To study the biodistribution of DNA origami in zebrafish and to determine differences in biodistribution based on the coating of the nanostructure using oligolysine PEG copolymers zebrafish embryos 2dpf were injected with coated or uncoated DNA origami wireframe nanosheets without xxx targeting molecule but containing a detection fluorophores. post treatment the embryos were disassociated into a single cell suspension and sorted through FACS for embryos containing nanostructures fluorophores. 10X sc libraries were generated from the sorted embryos and sequenced to establish cell type predictions for cells with coated and uncoated structures respectively.,ENA FIRST PUBLIC:2024 09 30|ENA LAST UPDATE:2024 09 30,,Protocols: Embryos 48 hpf were disassociated into a single cell suspension. Four hours post injections around 200 Tgfli1:EGFP embryos were euthanized in 0.02% tricaine. The tissue was dissociated with harsh pipetting in 0.25% trypsin EDTA GIBCO and 100mg/mL collagenase Sigma Aldrich for 3 min at 30oC followed by resuspension in DMEM 10% FBS. The cells were then pelleted at 700xg for 5 min washed once in 1X PBS filtered through a 40 μm cell strainer MERCK and stained with 1mM DAPI solution Abcam. TS+ cells were FACS sorted on a FACSARIA III with BD FACSDIVA software v. 9.0.1 BD Biosciences. Zebrafish D. rerio were maintained and bred in compliance with Swedish legislation on animal welfare regulations approved by Stockholms djurförsöksetiska nämnd. Each zebrafish embryo Tgfli1:EGFP was microinjected with 4 nl of the sample. The samples for injections were prepared at a final concentration of 100 nM in 1x PBS effective dose estimated to around 4 6 nM. Embryos at 48 hpf were anesthetized using 0.1% tricaine MS222 transferred onto an agarose surface placed on their ventral side and injected into the common cardinal vein Duct of Cuvier. scRNAseq libraries were generated from the sorted cells using 10X Genomics Chromium Single Cell 3ʹ Reagent Kits v3.1 per manufacturer's instructions targeting 8000 cells. The cDNA libraries were amplified using 10X Genomics Chromium Single Cell 3ʹ Reagent Kits v3.1 with 14 PCR cycles.,Coated,SAMEA114641726,KIS,ENA first public:2024 09 30|External Id:SAMEA114641726|INSDC center name:KIS|INSDC last update:2023 11 20T16:06:50Z|INSDC status:public|Submitter Id:E MTAB 13554:Coated|age:2|broker name:ArrayExpress|collection date:not collected|common name:zebrafish|developmental stage:embryo stage|geographic location country and/or sea:not collected|isolate:not applicable|organism part:whole organism|sample name:E MTAB 13554:Coated|scientific name:Danio rerio|sex:not available|strain:transgenic lines Tgfli1:EGFPy1,,,,,,,,,10X genomics libraries for scRNA seq of zebrafish embryos 2dpf treated with uncoated and oligolysine PEG copolymer coated DNA origami wireframe nanosheets,E MTAB 13554:Coated p,Coated p,10X genomics libraries for scRNA seq of zebrafish embryos 2dpf treated with uncoated and oligolysine PEG copolymer coated DNA origami wireframe nanosheets,Embryos 48 hpf were disassociated into a single cell suspension. Four hours post injections around 200 Tgfli1:EGFP embryos were euthanized in 0.02% tricaine. The tissue was dissociated with harsh pipetting in 0.25% trypsin EDTA GIBCO and 100mg/mL collagenase Sigma Aldrich for 3 min at 30oC followed by resuspension in DMEM 10% FBS. The cells were then pelleted at 700xg for 5 min washed once in 1X PBS filtered through a 40 μm cell strainer MERCK and stained with 1mM DAPI solution Abcam. TS+ cells were FACS sorted on a FACSARIA III with BD FACSDIVA software v. 9.0.1 BD Biosciences. Zebrafish D. rerio were maintained and bred in compliance with Swedish legislation on animal welfare regulations approved by Stockholms djurförsöksetiska nämnd. Each zebrafish embryo Tgfli1:EGFP was microinjected with 4 nl of the sample. The samples for injections were prepared at a final concentration of 100 nM in 1x PBS effective dose estimated to around 4 6 nM. Embryos at 48 hpf were anesthetized using 0.1% tricaine MS222 transferred onto an agarose surface placed on their ventral side and injected into the common cardinal vein Duct of Cuvier. scRNAseq libraries were generated from the sorted cells using 10X Genomics Chromium Single Cell 3ʹ Reagent Kits v3.1 per manufacturer's instructions targeting 8000 cells. The cDNA libraries were amplified using 10X Genomics Chromium Single Cell 3ʹ Reagent Kits v3.1 with 14 PCR cycles.,,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,PolyA,PAIRED,ILLUMINA,NextSeq 550,,ERP155237,NextSeq 550 paired end sequencing; 10X genomics libraries for scRNA seq of zebrafish embryos 2dpf treated with uncoated and oligolysine PEG copolymer coated DNA origami wireframe nanosheets,ENA FIRST PUBLIC:2024 09 30|ENA LAST UPDATE:2024 09 30,230324-Coated_S1_L001_I1_001.fastq.gz 230324-Coated_S1_L001_I2_001.fastq.gz 230324-Coated_S1_L001_R1_001.fastq.gz 230324-Coated_S1_L001_R2_001.fastq.gz,fastq fastq fastq fastq,9823147840.0,61394674.0,E MTAB 13554:230324 Coated S1 L001,0:10 1:10 2:30 3:110,A:1851113558;C:1521671335;G:1558230628;T:1822067428;N:331191,10,10,30,110,1851113558,1521671335,1558230628,1822067428,331191,ERX11683773,ERS17043893,ERA27417303,European Bioinformatics Institute|European Nucleotide Archive,European Bioinformatics Institute|European Nucleotide Archive,,,,,,,,,,,,B,,usable mapping rate,illumina,nextseq,unknown,poly_a,unknown,sc,single_cell_droplet,10x,,United Kingdom,2023-11-20,Hatching,Embryo,Whole Organism,All anatomical structures
15013,ERR12306833,ERX11683774,ERS17043894,ERP155237,PRJEB70303,10X genomics libraries for scRNA seq of zebrafish embryos 2dpf treated with uncoated and oligolysine PEG copolymer coated DNA origami wireframe nanosheets,E-MTAB-13554,Transcriptome Analysis,To study the biodistribution of DNA origami in zebrafish and to determine differences in biodistribution based on the coating of the nanostructure using oligolysine PEG copolymers zebrafish embryos 2dpf were injected with coated or uncoated DNA origami wireframe nanosheets without xxx targeting molecule but containing a detection fluorophores. post treatment the embryos were disassociated into a single cell suspension and sorted through FACS for embryos containing nanostructures fluorophores. 10X sc libraries were generated from the sorted embryos and sequenced to establish cell type predictions for cells with coated and uncoated structures respectively.,ENA FIRST PUBLIC:2024 09 30|ENA LAST UPDATE:2024 09 30,,Protocols: Embryos 48 hpf were disassociated into a single cell suspension. Four hours post injections around 200 Tgfli1:EGFP embryos were euthanized in 0.02% tricaine. The tissue was dissociated with harsh pipetting in 0.25% trypsin EDTA GIBCO and 100mg/mL collagenase Sigma Aldrich for 3 min at 30oC followed by resuspension in DMEM 10% FBS. The cells were then pelleted at 700xg for 5 min washed once in 1X PBS filtered through a 40 μm cell strainer MERCK and stained with 1mM DAPI solution Abcam. TS+ cells were FACS sorted on a FACSARIA III with BD FACSDIVA software v. 9.0.1 BD Biosciences. Zebrafish D. rerio were maintained and bred in compliance with Swedish legislation on animal welfare regulations approved by Stockholms djurförsöksetiska nämnd. Each zebrafish embryo Tgfli1:EGFP was microinjected with 4 nl of the sample. The samples for injections were prepared at a final concentration of 100 nM in 1x PBS effective dose estimated to around 4 6 nM. Embryos at 48 hpf were anesthetized using 0.1% tricaine MS222 transferred onto an agarose surface placed on their ventral side and injected into the common cardinal vein Duct of Cuvier. scRNAseq libraries were generated from the sorted cells using 10X Genomics Chromium Single Cell 3ʹ Reagent Kits v3.1 per manufacturer's instructions targeting 8000 cells. The cDNA libraries were amplified using 10X Genomics Chromium Single Cell 3ʹ Reagent Kits v3.1 with 14 PCR cycles.,Uncoated,SAMEA114641727,KIS,ENA first public:2024 09 30|External Id:SAMEA114641727|INSDC center name:KIS|INSDC last update:2023 11 20T16:06:50Z|INSDC status:public|Submitter Id:E MTAB 13554:Uncoated|age:2|broker name:ArrayExpress|collection date:not collected|common name:zebrafish|developmental stage:embryo stage|geographic location country and/or sea:not collected|isolate:not applicable|organism part:whole organism|sample name:E MTAB 13554:Uncoated|scientific name:Danio rerio|sex:not available|strain:transgenic lines Tgfli1:EGFPy1,,,,,,,,,10X genomics libraries for scRNA seq of zebrafish embryos 2dpf treated with uncoated and oligolysine PEG copolymer coated DNA origami wireframe nanosheets,E MTAB 13554:Uncoated p,Uncoated p,10X genomics libraries for scRNA seq of zebrafish embryos 2dpf treated with uncoated and oligolysine PEG copolymer coated DNA origami wireframe nanosheets,Embryos 48 hpf were disassociated into a single cell suspension. Four hours post injections around 200 Tgfli1:EGFP embryos were euthanized in 0.02% tricaine. The tissue was dissociated with harsh pipetting in 0.25% trypsin EDTA GIBCO and 100mg/mL collagenase Sigma Aldrich for 3 min at 30oC followed by resuspension in DMEM 10% FBS. The cells were then pelleted at 700xg for 5 min washed once in 1X PBS filtered through a 40 μm cell strainer MERCK and stained with 1mM DAPI solution Abcam. TS+ cells were FACS sorted on a FACSARIA III with BD FACSDIVA software v. 9.0.1 BD Biosciences. Zebrafish D. rerio were maintained and bred in compliance with Swedish legislation on animal welfare regulations approved by Stockholms djurförsöksetiska nämnd. Each zebrafish embryo Tgfli1:EGFP was microinjected with 4 nl of the sample. The samples for injections were prepared at a final concentration of 100 nM in 1x PBS effective dose estimated to around 4 6 nM. Embryos at 48 hpf were anesthetized using 0.1% tricaine MS222 transferred onto an agarose surface placed on their ventral side and injected into the common cardinal vein Duct of Cuvier. scRNAseq libraries were generated from the sorted cells using 10X Genomics Chromium Single Cell 3ʹ Reagent Kits v3.1 per manufacturer's instructions targeting 8000 cells. The cDNA libraries were amplified using 10X Genomics Chromium Single Cell 3ʹ Reagent Kits v3.1 with 14 PCR cycles.,,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,PolyA,PAIRED,ILLUMINA,NextSeq 550,,ERP155237,NextSeq 550 paired end sequencing; 10X genomics libraries for scRNA seq of zebrafish embryos 2dpf treated with uncoated and oligolysine PEG copolymer coated DNA origami wireframe nanosheets,ENA FIRST PUBLIC:2024 09 30|ENA LAST UPDATE:2024 09 30,230324-Uncoated_S2_L001_I1_001.fastq.gz 230324-Uncoated_S2_L001_I2_001.fastq.gz 230324-Uncoated_S2_L001_R1_001.fastq.gz 230324-Uncoated_S2_L001_R2_001.fastq.gz,fastq fastq fastq fastq,8871716800.0,55448230.0,E MTAB 13554:230324 Uncoated S2 L001,0:10 1:10 2:30 3:110,A:1718985845;C:1319560520;G:1372197713;T:1688254655;N:306567,10,10,30,110,1718985845,1319560520,1372197713,1688254655,306567,ERX11683774,ERS17043894,ERA27417303,European Bioinformatics Institute|European Nucleotide Archive,European Bioinformatics Institute|European Nucleotide Archive,,,,,,,,,,,,B,,usable mapping rate,illumina,nextseq,unknown,poly_a,unknown,sc,single_cell_droplet,10x,,United Kingdom,2023-11-20,Hatching,Embryo,Whole Organism,All anatomical structures
15014,ERR12306830,ERX11683774,ERS17043894,ERP155237,PRJEB70303,10X genomics libraries for scRNA seq of zebrafish embryos 2dpf treated with uncoated and oligolysine PEG copolymer coated DNA origami wireframe nanosheets,E-MTAB-13554,Transcriptome Analysis,To study the biodistribution of DNA origami in zebrafish and to determine differences in biodistribution based on the coating of the nanostructure using oligolysine PEG copolymers zebrafish embryos 2dpf were injected with coated or uncoated DNA origami wireframe nanosheets without xxx targeting molecule but containing a detection fluorophores. post treatment the embryos were disassociated into a single cell suspension and sorted through FACS for embryos containing nanostructures fluorophores. 10X sc libraries were generated from the sorted embryos and sequenced to establish cell type predictions for cells with coated and uncoated structures respectively.,ENA FIRST PUBLIC:2024 09 30|ENA LAST UPDATE:2024 09 30,,Protocols: Embryos 48 hpf were disassociated into a single cell suspension. Four hours post injections around 200 Tgfli1:EGFP embryos were euthanized in 0.02% tricaine. The tissue was dissociated with harsh pipetting in 0.25% trypsin EDTA GIBCO and 100mg/mL collagenase Sigma Aldrich for 3 min at 30oC followed by resuspension in DMEM 10% FBS. The cells were then pelleted at 700xg for 5 min washed once in 1X PBS filtered through a 40 μm cell strainer MERCK and stained with 1mM DAPI solution Abcam. TS+ cells were FACS sorted on a FACSARIA III with BD FACSDIVA software v. 9.0.1 BD Biosciences. Zebrafish D. rerio were maintained and bred in compliance with Swedish legislation on animal welfare regulations approved by Stockholms djurförsöksetiska nämnd. Each zebrafish embryo Tgfli1:EGFP was microinjected with 4 nl of the sample. The samples for injections were prepared at a final concentration of 100 nM in 1x PBS effective dose estimated to around 4 6 nM. Embryos at 48 hpf were anesthetized using 0.1% tricaine MS222 transferred onto an agarose surface placed on their ventral side and injected into the common cardinal vein Duct of Cuvier. scRNAseq libraries were generated from the sorted cells using 10X Genomics Chromium Single Cell 3ʹ Reagent Kits v3.1 per manufacturer's instructions targeting 8000 cells. The cDNA libraries were amplified using 10X Genomics Chromium Single Cell 3ʹ Reagent Kits v3.1 with 14 PCR cycles.,Uncoated,SAMEA114641727,KIS,ENA first public:2024 09 30|External Id:SAMEA114641727|INSDC center name:KIS|INSDC last update:2023 11 20T16:06:50Z|INSDC status:public|Submitter Id:E MTAB 13554:Uncoated|age:2|broker name:ArrayExpress|collection date:not collected|common name:zebrafish|developmental stage:embryo stage|geographic location country and/or sea:not collected|isolate:not applicable|organism part:whole organism|sample name:E MTAB 13554:Uncoated|scientific name:Danio rerio|sex:not available|strain:transgenic lines Tgfli1:EGFPy1,,,,,,,,,10X genomics libraries for scRNA seq of zebrafish embryos 2dpf treated with uncoated and oligolysine PEG copolymer coated DNA origami wireframe nanosheets,E MTAB 13554:Uncoated p,Uncoated p,10X genomics libraries for scRNA seq of zebrafish embryos 2dpf treated with uncoated and oligolysine PEG copolymer coated DNA origami wireframe nanosheets,Embryos 48 hpf were disassociated into a single cell suspension. Four hours post injections around 200 Tgfli1:EGFP embryos were euthanized in 0.02% tricaine. The tissue was dissociated with harsh pipetting in 0.25% trypsin EDTA GIBCO and 100mg/mL collagenase Sigma Aldrich for 3 min at 30oC followed by resuspension in DMEM 10% FBS. The cells were then pelleted at 700xg for 5 min washed once in 1X PBS filtered through a 40 μm cell strainer MERCK and stained with 1mM DAPI solution Abcam. TS+ cells were FACS sorted on a FACSARIA III with BD FACSDIVA software v. 9.0.1 BD Biosciences. Zebrafish D. rerio were maintained and bred in compliance with Swedish legislation on animal welfare regulations approved by Stockholms djurförsöksetiska nämnd. Each zebrafish embryo Tgfli1:EGFP was microinjected with 4 nl of the sample. The samples for injections were prepared at a final concentration of 100 nM in 1x PBS effective dose estimated to around 4 6 nM. Embryos at 48 hpf were anesthetized using 0.1% tricaine MS222 transferred onto an agarose surface placed on their ventral side and injected into the common cardinal vein Duct of Cuvier. scRNAseq libraries were generated from the sorted cells using 10X Genomics Chromium Single Cell 3ʹ Reagent Kits v3.1 per manufacturer's instructions targeting 8000 cells. The cDNA libraries were amplified using 10X Genomics Chromium Single Cell 3ʹ Reagent Kits v3.1 with 14 PCR cycles.,,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,PolyA,PAIRED,ILLUMINA,NextSeq 550,,ERP155237,NextSeq 550 paired end sequencing; 10X genomics libraries for scRNA seq of zebrafish embryos 2dpf treated with uncoated and oligolysine PEG copolymer coated DNA origami wireframe nanosheets,ENA FIRST PUBLIC:2024 09 30|ENA LAST UPDATE:2024 09 30,230324-Uncoated_S2_L003_I1_001.fastq.gz 230324-Uncoated_S2_L003_I2_001.fastq.gz 230324-Uncoated_S2_L003_R1_001.fastq.gz 230324-Uncoated_S2_L003_R2_001.fastq.gz,fastq fastq fastq fastq,9075478720.0,56721742.0,E MTAB 13554:230324 Uncoated S2 L003,0:10 1:10 2:30 3:110,A:1758677834;C:1349774831;G:1404463433;T:1725994897;N:480625,10,10,30,110,1758677834,1349774831,1404463433,1725994897,480625,ERX11683774,ERS17043894,ERA27417303,European Bioinformatics Institute|European Nucleotide Archive,European Bioinformatics Institute|European Nucleotide Archive,,,,,,,,,,,,B,,usable mapping rate,illumina,nextseq,unknown,poly_a,unknown,sc,single_cell_droplet,10x,,United Kingdom,2023-11-20,Hatching,Embryo,Whole Organism,All anatomical structures
15015,ERR12306831,ERX11683774,ERS17043894,ERP155237,PRJEB70303,10X genomics libraries for scRNA seq of zebrafish embryos 2dpf treated with uncoated and oligolysine PEG copolymer coated DNA origami wireframe nanosheets,E-MTAB-13554,Transcriptome Analysis,To study the biodistribution of DNA origami in zebrafish and to determine differences in biodistribution based on the coating of the nanostructure using oligolysine PEG copolymers zebrafish embryos 2dpf were injected with coated or uncoated DNA origami wireframe nanosheets without xxx targeting molecule but containing a detection fluorophores. post treatment the embryos were disassociated into a single cell suspension and sorted through FACS for embryos containing nanostructures fluorophores. 10X sc libraries were generated from the sorted embryos and sequenced to establish cell type predictions for cells with coated and uncoated structures respectively.,ENA FIRST PUBLIC:2024 09 30|ENA LAST UPDATE:2024 09 30,,Protocols: Embryos 48 hpf were disassociated into a single cell suspension. Four hours post injections around 200 Tgfli1:EGFP embryos were euthanized in 0.02% tricaine. The tissue was dissociated with harsh pipetting in 0.25% trypsin EDTA GIBCO and 100mg/mL collagenase Sigma Aldrich for 3 min at 30oC followed by resuspension in DMEM 10% FBS. The cells were then pelleted at 700xg for 5 min washed once in 1X PBS filtered through a 40 μm cell strainer MERCK and stained with 1mM DAPI solution Abcam. TS+ cells were FACS sorted on a FACSARIA III with BD FACSDIVA software v. 9.0.1 BD Biosciences. Zebrafish D. rerio were maintained and bred in compliance with Swedish legislation on animal welfare regulations approved by Stockholms djurförsöksetiska nämnd. Each zebrafish embryo Tgfli1:EGFP was microinjected with 4 nl of the sample. The samples for injections were prepared at a final concentration of 100 nM in 1x PBS effective dose estimated to around 4 6 nM. Embryos at 48 hpf were anesthetized using 0.1% tricaine MS222 transferred onto an agarose surface placed on their ventral side and injected into the common cardinal vein Duct of Cuvier. scRNAseq libraries were generated from the sorted cells using 10X Genomics Chromium Single Cell 3ʹ Reagent Kits v3.1 per manufacturer's instructions targeting 8000 cells. The cDNA libraries were amplified using 10X Genomics Chromium Single Cell 3ʹ Reagent Kits v3.1 with 14 PCR cycles.,Uncoated,SAMEA114641727,KIS,ENA first public:2024 09 30|External Id:SAMEA114641727|INSDC center name:KIS|INSDC last update:2023 11 20T16:06:50Z|INSDC status:public|Submitter Id:E MTAB 13554:Uncoated|age:2|broker name:ArrayExpress|collection date:not collected|common name:zebrafish|developmental stage:embryo stage|geographic location country and/or sea:not collected|isolate:not applicable|organism part:whole organism|sample name:E MTAB 13554:Uncoated|scientific name:Danio rerio|sex:not available|strain:transgenic lines Tgfli1:EGFPy1,,,,,,,,,10X genomics libraries for scRNA seq of zebrafish embryos 2dpf treated with uncoated and oligolysine PEG copolymer coated DNA origami wireframe nanosheets,E MTAB 13554:Uncoated p,Uncoated p,10X genomics libraries for scRNA seq of zebrafish embryos 2dpf treated with uncoated and oligolysine PEG copolymer coated DNA origami wireframe nanosheets,Embryos 48 hpf were disassociated into a single cell suspension. Four hours post injections around 200 Tgfli1:EGFP embryos were euthanized in 0.02% tricaine. The tissue was dissociated with harsh pipetting in 0.25% trypsin EDTA GIBCO and 100mg/mL collagenase Sigma Aldrich for 3 min at 30oC followed by resuspension in DMEM 10% FBS. The cells were then pelleted at 700xg for 5 min washed once in 1X PBS filtered through a 40 μm cell strainer MERCK and stained with 1mM DAPI solution Abcam. TS+ cells were FACS sorted on a FACSARIA III with BD FACSDIVA software v. 9.0.1 BD Biosciences. Zebrafish D. rerio were maintained and bred in compliance with Swedish legislation on animal welfare regulations approved by Stockholms djurförsöksetiska nämnd. Each zebrafish embryo Tgfli1:EGFP was microinjected with 4 nl of the sample. The samples for injections were prepared at a final concentration of 100 nM in 1x PBS effective dose estimated to around 4 6 nM. Embryos at 48 hpf were anesthetized using 0.1% tricaine MS222 transferred onto an agarose surface placed on their ventral side and injected into the common cardinal vein Duct of Cuvier. scRNAseq libraries were generated from the sorted cells using 10X Genomics Chromium Single Cell 3ʹ Reagent Kits v3.1 per manufacturer's instructions targeting 8000 cells. The cDNA libraries were amplified using 10X Genomics Chromium Single Cell 3ʹ Reagent Kits v3.1 with 14 PCR cycles.,,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,PolyA,PAIRED,ILLUMINA,NextSeq 550,,ERP155237,NextSeq 550 paired end sequencing; 10X genomics libraries for scRNA seq of zebrafish embryos 2dpf treated with uncoated and oligolysine PEG copolymer coated DNA origami wireframe nanosheets,ENA FIRST PUBLIC:2024 09 30|ENA LAST UPDATE:2024 09 30,230324-Uncoated_S2_L002_I1_001.fastq.gz 230324-Uncoated_S2_L002_I2_001.fastq.gz 230324-Uncoated_S2_L002_R1_001.fastq.gz 230324-Uncoated_S2_L002_R2_001.fastq.gz,fastq fastq fastq fastq,8705210880.0,54407568.0,E MTAB 13554:230324 Uncoated S2 L002,0:10 1:10 2:30 3:110,A:1687330583;C:1295837902;G:1345522657;T:1655826422;N:314916,10,10,30,110,1687330583,1295837902,1345522657,1655826422,314916,ERX11683774,ERS17043894,ERA27417303,European Bioinformatics Institute|European Nucleotide Archive,European Bioinformatics Institute|European Nucleotide Archive,,,,,,,,,,,,B,,usable mapping rate,illumina,nextseq,unknown,poly_a,unknown,sc,single_cell_droplet,10x,,United Kingdom,2023-11-20,Hatching,Embryo,Whole Organism,All anatomical structures
15016,ERR12306827,ERX11683774,ERS17043894,ERP155237,PRJEB70303,10X genomics libraries for scRNA seq of zebrafish embryos 2dpf treated with uncoated and oligolysine PEG copolymer coated DNA origami wireframe nanosheets,E-MTAB-13554,Transcriptome Analysis,To study the biodistribution of DNA origami in zebrafish and to determine differences in biodistribution based on the coating of the nanostructure using oligolysine PEG copolymers zebrafish embryos 2dpf were injected with coated or uncoated DNA origami wireframe nanosheets without xxx targeting molecule but containing a detection fluorophores. post treatment the embryos were disassociated into a single cell suspension and sorted through FACS for embryos containing nanostructures fluorophores. 10X sc libraries were generated from the sorted embryos and sequenced to establish cell type predictions for cells with coated and uncoated structures respectively.,ENA FIRST PUBLIC:2024 09 30|ENA LAST UPDATE:2024 09 30,,Protocols: Embryos 48 hpf were disassociated into a single cell suspension. Four hours post injections around 200 Tgfli1:EGFP embryos were euthanized in 0.02% tricaine. The tissue was dissociated with harsh pipetting in 0.25% trypsin EDTA GIBCO and 100mg/mL collagenase Sigma Aldrich for 3 min at 30oC followed by resuspension in DMEM 10% FBS. The cells were then pelleted at 700xg for 5 min washed once in 1X PBS filtered through a 40 μm cell strainer MERCK and stained with 1mM DAPI solution Abcam. TS+ cells were FACS sorted on a FACSARIA III with BD FACSDIVA software v. 9.0.1 BD Biosciences. Zebrafish D. rerio were maintained and bred in compliance with Swedish legislation on animal welfare regulations approved by Stockholms djurförsöksetiska nämnd. Each zebrafish embryo Tgfli1:EGFP was microinjected with 4 nl of the sample. The samples for injections were prepared at a final concentration of 100 nM in 1x PBS effective dose estimated to around 4 6 nM. Embryos at 48 hpf were anesthetized using 0.1% tricaine MS222 transferred onto an agarose surface placed on their ventral side and injected into the common cardinal vein Duct of Cuvier. scRNAseq libraries were generated from the sorted cells using 10X Genomics Chromium Single Cell 3ʹ Reagent Kits v3.1 per manufacturer's instructions targeting 8000 cells. The cDNA libraries were amplified using 10X Genomics Chromium Single Cell 3ʹ Reagent Kits v3.1 with 14 PCR cycles.,Uncoated,SAMEA114641727,KIS,ENA first public:2024 09 30|External Id:SAMEA114641727|INSDC center name:KIS|INSDC last update:2023 11 20T16:06:50Z|INSDC status:public|Submitter Id:E MTAB 13554:Uncoated|age:2|broker name:ArrayExpress|collection date:not collected|common name:zebrafish|developmental stage:embryo stage|geographic location country and/or sea:not collected|isolate:not applicable|organism part:whole organism|sample name:E MTAB 13554:Uncoated|scientific name:Danio rerio|sex:not available|strain:transgenic lines Tgfli1:EGFPy1,,,,,,,,,10X genomics libraries for scRNA seq of zebrafish embryos 2dpf treated with uncoated and oligolysine PEG copolymer coated DNA origami wireframe nanosheets,E MTAB 13554:Uncoated p,Uncoated p,10X genomics libraries for scRNA seq of zebrafish embryos 2dpf treated with uncoated and oligolysine PEG copolymer coated DNA origami wireframe nanosheets,Embryos 48 hpf were disassociated into a single cell suspension. Four hours post injections around 200 Tgfli1:EGFP embryos were euthanized in 0.02% tricaine. The tissue was dissociated with harsh pipetting in 0.25% trypsin EDTA GIBCO and 100mg/mL collagenase Sigma Aldrich for 3 min at 30oC followed by resuspension in DMEM 10% FBS. The cells were then pelleted at 700xg for 5 min washed once in 1X PBS filtered through a 40 μm cell strainer MERCK and stained with 1mM DAPI solution Abcam. TS+ cells were FACS sorted on a FACSARIA III with BD FACSDIVA software v. 9.0.1 BD Biosciences. Zebrafish D. rerio were maintained and bred in compliance with Swedish legislation on animal welfare regulations approved by Stockholms djurförsöksetiska nämnd. Each zebrafish embryo Tgfli1:EGFP was microinjected with 4 nl of the sample. The samples for injections were prepared at a final concentration of 100 nM in 1x PBS effective dose estimated to around 4 6 nM. Embryos at 48 hpf were anesthetized using 0.1% tricaine MS222 transferred onto an agarose surface placed on their ventral side and injected into the common cardinal vein Duct of Cuvier. scRNAseq libraries were generated from the sorted cells using 10X Genomics Chromium Single Cell 3ʹ Reagent Kits v3.1 per manufacturer's instructions targeting 8000 cells. The cDNA libraries were amplified using 10X Genomics Chromium Single Cell 3ʹ Reagent Kits v3.1 with 14 PCR cycles.,,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,PolyA,PAIRED,ILLUMINA,NextSeq 550,,ERP155237,NextSeq 550 paired end sequencing; 10X genomics libraries for scRNA seq of zebrafish embryos 2dpf treated with uncoated and oligolysine PEG copolymer coated DNA origami wireframe nanosheets,ENA FIRST PUBLIC:2024 09 30|ENA LAST UPDATE:2024 09 30,230324-Uncoated_S2_L004_I1_001.fastq.gz 230324-Uncoated_S2_L004_I2_001.fastq.gz 230324-Uncoated_S2_L004_R1_001.fastq.gz 230324-Uncoated_S2_L004_R2_001.fastq.gz,fastq fastq fastq fastq,8846203360.0,55288771.0,E MTAB 13554:230324 Uncoated S2 L004,0:10 1:10 2:30 3:110,A:1715100047;C:1316853239;G:1367208350;T:1682248092;N:355082,10,10,30,110,1715100047,1316853239,1367208350,1682248092,355082,ERX11683774,ERS17043894,ERA27417303,European Bioinformatics Institute|European Nucleotide Archive,European Bioinformatics Institute|European Nucleotide Archive,,,,,,,,,,,,B,,usable mapping rate,illumina,nextseq,unknown,poly_a,unknown,sc,single_cell_droplet,10x,,United Kingdom,2023-11-20,Hatching,Embryo,Whole Organism,All anatomical structures
36032,SRR33405556,SRX28646822,SRS24917431,SRP582714,PRJNA1257950,Zebrafish Timothy syndrome knock in model,PRJNA1257950,Other,Characterization of transcriptomic changes in anterior tissue of zebrafish larvae with mutation in cacna1c to model Timothy sundrome.,,,,,mut2 d2,,strain:ABC|age:2 dpf|collection date:2024 06|geo loc name:USA: Maryland Bethesda|sex:n/a|tissue:Anterior to swimbladder|genotype:mut|replicate:mut 2 dpf replicate 2|BioSampleModel:Model organism or animal,,,,,,,,,RNA Seq of zebrafish TS2 mutant,mut2 d2,mut2 d2,TruSeq library,,,RNA-Seq,TRANSCRIPTOMIC,PolyA,PAIRED,ILLUMINA,Illumina NovaSeq 6000,,SRP582714,,,y680_mut-2_S6_R1_001.fastq.gz y680_mut-2_S6_R2_001.fastq.gz,fastq fastq,13048391588.0,65176490.0,y680 mut 2 S6 R1 001.fastq.gz,0:100.12 1:100.08,A:3382606790;C:3166963239;G:3249911967;T:3247119183;N:1790409,100,100,,,3382606790,3166963239,3249911967,3247119183,1790409,SRX28646822,SRS24917431,SRA2122396,NICHD|DDB,NICHD,,,,,,,,,,,,B,B,biological fallback assumption,illumina,novaseq_era,unknown,poly_a,trueseq,bulk,unknown,unknown,,United States,2025-05-01,Hatching,Embryo,Undetermined,Embryo Imprecise
36033,SRR33405557,SRX28646821,SRS24917430,SRP582714,PRJNA1257950,Zebrafish Timothy syndrome knock in model,PRJNA1257950,Other,Characterization of transcriptomic changes in anterior tissue of zebrafish larvae with mutation in cacna1c to model Timothy sundrome.,,,,,mut1 d2,,strain:ABC|age:2 dpf|collection date:2024 06|geo loc name:USA: Maryland Bethesda|sex:n/a|tissue:Anterior to swimbladder|genotype:mut|replicate:mut 2 dpf replicate 1|BioSampleModel:Model organism or animal,,,,,,,,,RNA Seq of zebrafish TS2 mutant,mut1 d2,mut1 d2,TruSeq library,,,RNA-Seq,TRANSCRIPTOMIC,PolyA,PAIRED,ILLUMINA,Illumina NovaSeq 6000,,SRP582714,,,y680_mut-1_S5_R1_001.fastq.gz y680_mut-1_S5_R2_001.fastq.gz,fastq fastq,11244387522.0,56145229.0,y680 mut 1 S5 R1 001.fastq.gz,0:100.15 1:100.12,A:2893088657;C:2737659501;G:2830491662;T:2781546759;N:1600943,100,100,,,2893088657,2737659501,2830491662,2781546759,1600943,SRX28646821,SRS24917430,SRA2122396,NICHD|DDB,NICHD,,,,,,,,,,,,B,B,biological fallback assumption,illumina,novaseq_era,unknown,poly_a,trueseq,bulk,unknown,unknown,,United States,2025-05-01,Hatching,Embryo,Undetermined,Embryo Imprecise
36034,SRR33405558,SRX28646820,SRS24917429,SRP582714,PRJNA1257950,Zebrafish Timothy syndrome knock in model,PRJNA1257950,Other,Characterization of transcriptomic changes in anterior tissue of zebrafish larvae with mutation in cacna1c to model Timothy sundrome.,,,,,het4 d2,,strain:ABC|age:2 dpf|collection date:2024 06|geo loc name:USA: Maryland Bethesda|sex:n/a|tissue:Anterior to swimbladder|genotype:het|replicate:het 2 dpf replicate 4|BioSampleModel:Model organism or animal,,,,,,,,,RNA Seq of zebrafish TS2 mutant,het4 d2,het4 d2,TruSeq library,,,RNA-Seq,TRANSCRIPTOMIC,PolyA,PAIRED,ILLUMINA,Illumina NovaSeq 6000,,SRP582714,,,y680_het-4_S4_R1_001.fastq.gz y680_het-4_S4_R2_001.fastq.gz,fastq fastq,7488095202.0,37372687.0,y680 het 4 S4 R1 001.fastq.gz,0:100.20 1:100.16,A:1931359037;C:1813371312;G:1880760315;T:1861860377;N:744161,100,100,,,1931359037,1813371312,1880760315,1861860377,744161,SRX28646820,SRS24917429,SRA2122396,NICHD|DDB,NICHD,,,,,,,,,,,,B,B,biological fallback assumption,illumina,novaseq_era,unknown,poly_a,trueseq,bulk,unknown,unknown,,United States,2025-05-01,Hatching,Embryo,Undetermined,Embryo Imprecise
36035,SRR33405559,SRX28646819,SRS24917428,SRP582714,PRJNA1257950,Zebrafish Timothy syndrome knock in model,PRJNA1257950,Other,Characterization of transcriptomic changes in anterior tissue of zebrafish larvae with mutation in cacna1c to model Timothy sundrome.,,,,,het3 d2,,strain:ABC|age:2 dpf|collection date:2024 06|geo loc name:USA: Maryland Bethesda|sex:n/a|tissue:Anterior to swimbladder|genotype:het|replicate:het 2 dpf replicate 3|BioSampleModel:Model organism or animal,,,,,,,,,RNA Seq of zebrafish TS2 mutant,het3 d2,het3 d2,TruSeq library,,,RNA-Seq,TRANSCRIPTOMIC,PolyA,PAIRED,ILLUMINA,Illumina NovaSeq 6000,,SRP582714,,,y680_het-3_S3_R1_001.fastq.gz y680_het-3_S3_R2_001.fastq.gz,fastq fastq,13441009956.0,67072919.0,y680 het 3 S3 R1 001.fastq.gz,0:100.22 1:100.18,A:3484306615;C:3249847841;G:3351167195;T:3353988961;N:1699344,100,100,,,3484306615,3249847841,3351167195,3353988961,1699344,SRX28646819,SRS24917428,SRA2122396,NICHD|DDB,NICHD,,,,,,,,,,,,B,B,biological fallback assumption,illumina,novaseq_era,unknown,poly_a,trueseq,bulk,unknown,unknown,,United States,2025-05-01,Hatching,Embryo,Undetermined,Embryo Imprecise
36036,SRR33405560,SRX28646818,SRS24917427,SRP582714,PRJNA1257950,Zebrafish Timothy syndrome knock in model,PRJNA1257950,Other,Characterization of transcriptomic changes in anterior tissue of zebrafish larvae with mutation in cacna1c to model Timothy sundrome.,,,,,het2 d2,,strain:ABC|age:2 dpf|collection date:2024 06|geo loc name:USA: Maryland Bethesda|sex:n/a|tissue:Anterior to swimbladder|genotype:het|replicate:het 2 dpf replicate 2|BioSampleModel:Model organism or animal,,,,,,,,,RNA Seq of zebrafish TS2 mutant,het2 d2,het2 d2,TruSeq library,,,RNA-Seq,TRANSCRIPTOMIC,PolyA,PAIRED,ILLUMINA,Illumina NovaSeq 6000,,SRP582714,,,y680_het-2_S2_R1_001.fastq.gz y680_het-2_S2_R2_001.fastq.gz,fastq fastq,10048330828.0,50202819.0,y680 het 2 S2 R1 001.fastq.gz,0:100.10 1:100.05,A:2557623639;C:2473642678;G:2526118363;T:2490175343;N:770805,100,100,,,2557623639,2473642678,2526118363,2490175343,770805,SRX28646818,SRS24917427,SRA2122396,NICHD|DDB,NICHD,,,,,,,,,,,,B,B,biological fallback assumption,illumina,novaseq_era,unknown,poly_a,trueseq,bulk,unknown,unknown,,United States,2025-05-01,Hatching,Embryo,Undetermined,Embryo Imprecise
36037,SRR33405561,SRX28646817,SRS24917426,SRP582714,PRJNA1257950,Zebrafish Timothy syndrome knock in model,PRJNA1257950,Other,Characterization of transcriptomic changes in anterior tissue of zebrafish larvae with mutation in cacna1c to model Timothy sundrome.,,,,,het1 d2,,strain:ABC|age:2 dpf|collection date:2024 06|geo loc name:USA: Maryland Bethesda|sex:n/a|tissue:Anterior to swimbladder|genotype:het|replicate:het 2 dpf replicate 1|BioSampleModel:Model organism or animal,,,,,,,,,RNA Seq of zebrafish TS2 mutant,het1 d2,het1 d2,TruSeq library,,,RNA-Seq,TRANSCRIPTOMIC,PolyA,PAIRED,ILLUMINA,Illumina NovaSeq 6000,,SRP582714,,,y680_het-1_S1_R1_001.fastq.gz y680_het-1_S1_R2_001.fastq.gz,fastq fastq,21241485328.0,106063264.0,y680 het 1 S1 R1 001.fastq.gz,0:100.16 1:100.11,A:5428876256;C:5216202525;G:5316134708;T:5278092708;N:2179131,100,100,,,5428876256,5216202525,5316134708,5278092708,2179131,SRX28646817,SRS24917426,SRA2122396,NICHD|DDB,NICHD,,,,,,,,,,,,B,B,biological fallback assumption,illumina,novaseq_era,unknown,poly_a,trueseq,bulk,unknown,unknown,,United States,2025-05-01,Hatching,Embryo,Undetermined,Embryo Imprecise
36038,SRR33405562,SRX28646816,SRS24917425,SRP582714,PRJNA1257950,Zebrafish Timothy syndrome knock in model,PRJNA1257950,Other,Characterization of transcriptomic changes in anterior tissue of zebrafish larvae with mutation in cacna1c to model Timothy sundrome.,,,,,wt4 d2,,strain:ABC|age:2 dpf|collection date:2024 06|geo loc name:USA: Maryland Bethesda|sex:n/a|tissue:Anterior to swimbladder|genotype:WT|replicate:wt 2 dpf replicate 4|BioSampleModel:Model organism or animal,,,,,,,,,RNA Seq of zebrafish TS2 mutant,wt4 d2,wt4 d2,TruSeq library,,,RNA-Seq,TRANSCRIPTOMIC,PolyA,PAIRED,ILLUMINA,Illumina NovaSeq 6000,,SRP582714,,,y680-WT-4_S56_R1_001.fastq.gz y680-WT-4_S56_R2_001.fastq.gz,fastq fastq,2871774704.0,14403351.0,y680 WT 4 S56 R1 001.fastq.gz,0:99.62 1:99.76,A:738963438;C:693060627;G:725455885;T:712804013;N:1490741,99,99,,,738963438,693060627,725455885,712804013,1490741,SRX28646816,SRS24917425,SRA2122396,NICHD|DDB,NICHD,,,,,,,,,,,,B,B,biological fallback assumption,illumina,novaseq_era,unknown,poly_a,trueseq,bulk,unknown,unknown,,United States,2025-05-01,Hatching,Embryo,Undetermined,Embryo Imprecise
36040,SRR33405564,SRX28646814,SRS24917423,SRP582714,PRJNA1257950,Zebrafish Timothy syndrome knock in model,PRJNA1257950,Other,Characterization of transcriptomic changes in anterior tissue of zebrafish larvae with mutation in cacna1c to model Timothy sundrome.,,,,,wt3 d2,,strain:ABC|age:2 dpf|collection date:2024 06|geo loc name:USA: Maryland Bethesda|sex:n/a|tissue:Anterior to swimbladder|genotype:WT|replicate:wt 2 dpf replicate 3|BioSampleModel:Model organism or animal,,,,,,,,,RNA Seq of zebrafish TS2 mutant,wt3 d2,wt3 d2,TruSeq library,,,RNA-Seq,TRANSCRIPTOMIC,PolyA,PAIRED,ILLUMINA,Illumina NovaSeq 6000,,SRP582714,,,y680_WT-3_S11_R1_001.fastq.gz y680_WT-3_S11_R2_001.fastq.gz,fastq fastq,4644100900.0,23208636.0,y680 WT 3 S11 R1 001.fastq.gz,0:100.02 1:100.08,A:1208863635;C:1115875593;G:1183409664;T:1134173818;N:1778190,100,100,,,1208863635,1115875593,1183409664,1134173818,1778190,SRX28646814,SRS24917423,SRA2122396,NICHD|DDB,NICHD,,,,,,,,,,,,B,B,biological fallback assumption,illumina,novaseq_era,unknown,poly_a,trueseq,bulk,unknown,unknown,,United States,2025-05-01,Hatching,Embryo,Undetermined,Embryo Imprecise
36049,SRR33405573,SRX28646805,SRS24917414,SRP582714,PRJNA1257950,Zebrafish Timothy syndrome knock in model,PRJNA1257950,Other,Characterization of transcriptomic changes in anterior tissue of zebrafish larvae with mutation in cacna1c to model Timothy sundrome.,,,,,mut4 d2,,strain:ABC|age:2 dpf|collection date:2024 06|geo loc name:USA: Maryland Bethesda|sex:n/a|tissue:Anterior to swimbladder|genotype:mut|replicate:mut 2 dpf replicate 4|BioSampleModel:Model organism or animal,,,,,,,,,RNA Seq of zebrafish TS2 mutant,mut4 d2,mut4 d2,TruSeq library,,,RNA-Seq,TRANSCRIPTOMIC,PolyA,PAIRED,ILLUMINA,Illumina NovaSeq 6000,,SRP582714,,,y680_mut-4_S8_R1_001.fastq.gz y680_mut-4_S8_R2_001.fastq.gz,fastq fastq,6364246187.0,31799210.0,y680 mut 4 S8 R1 001.fastq.gz,0:100.05 1:100.09,A:1644746630;C:1536469430;G:1617907325;T:1562993280;N:2129522,100,100,,,1644746630,1536469430,1617907325,1562993280,2129522,SRX28646805,SRS24917414,SRA2122396,NICHD|DDB,NICHD,,,,,,,,,,,,B,B,biological fallback assumption,illumina,novaseq_era,unknown,poly_a,trueseq,bulk,unknown,unknown,,United States,2025-05-01,Hatching,Embryo,Undetermined,Embryo Imprecise
36050,SRR33405574,SRX28646804,SRS24917413,SRP582714,PRJNA1257950,Zebrafish Timothy syndrome knock in model,PRJNA1257950,Other,Characterization of transcriptomic changes in anterior tissue of zebrafish larvae with mutation in cacna1c to model Timothy sundrome.,,,,,mut3 d2,,strain:ABC|age:2 dpf|collection date:2024 06|geo loc name:USA: Maryland Bethesda|sex:n/a|tissue:Anterior to swimbladder|genotype:mut|replicate:mut 2 dpf replicate 3|BioSampleModel:Model organism or animal,,,,,,,,,RNA Seq of zebrafish TS2 mutant,mut3 d2,mut3 d2,TruSeq library,,,RNA-Seq,TRANSCRIPTOMIC,PolyA,PAIRED,ILLUMINA,Illumina NovaSeq 6000,,SRP582714,,,y680_mut-3_S7_R1_001.fastq.gz y680_mut-3_S7_R2_001.fastq.gz,fastq fastq,12769594212.0,63754551.0,y680 mut 3 S7 R1 001.fastq.gz,0:100.17 1:100.12,A:3270488183;C:3128646350;G:3212521095;T:3156861188;N:1077396,100,100,,,3270488183,3128646350,3212521095,3156861188,1077396,SRX28646804,SRS24917413,SRA2122396,NICHD|DDB,NICHD,,,,,,,,,,,,B,B,biological fallback assumption,illumina,novaseq_era,unknown,poly_a,trueseq,bulk,unknown,unknown,,United States,2025-05-01,Hatching,Embryo,Undetermined,Embryo Imprecise
36051,SRR33405575,SRX28646803,SRS24917412,SRP582714,PRJNA1257950,Zebrafish Timothy syndrome knock in model,PRJNA1257950,Other,Characterization of transcriptomic changes in anterior tissue of zebrafish larvae with mutation in cacna1c to model Timothy sundrome.,,,,,wt2 d2,,strain:ABC|age:2 dpf|collection date:2024 06|geo loc name:USA: Maryland Bethesda|sex:n/a|tissue:Anterior to swimbladder|genotype:WT|replicate:wt 2 dpf replicate 2|BioSampleModel:Model organism or animal,,,,,,,,,RNA Seq of zebrafish TS2 mutant,wt2 d2,wt2 d2,TruSeq library,,,RNA-Seq,TRANSCRIPTOMIC,PolyA,PAIRED,ILLUMINA,Illumina NovaSeq 6000,,SRP582714,,,y680-WT-2_S40_R1_001.fastq.gz y680-WT-2_S40_R2_001.fastq.gz,fastq fastq,7228768016.0,36426014.0,y680 WT 2 S40 R1 001.fastq.gz,0:99.18 1:99.27,A:1808349692;C:1804255213;G:1817770107;T:1795596464;N:2796540,99,99,,,1808349692,1804255213,1817770107,1795596464,2796540,SRX28646803,SRS24917412,SRA2122396,NICHD|DDB,NICHD,,,,,,,,,,,,B,B,biological fallback assumption,illumina,novaseq_era,unknown,poly_a,trueseq,bulk,unknown,unknown,,United States,2025-05-01,Hatching,Embryo,Undetermined,Embryo Imprecise
36052,SRR33405576,SRX28646802,SRS24917411,SRP582714,PRJNA1257950,Zebrafish Timothy syndrome knock in model,PRJNA1257950,Other,Characterization of transcriptomic changes in anterior tissue of zebrafish larvae with mutation in cacna1c to model Timothy sundrome.,,,,,wt1 d2,,strain:ABC|age:2 dpf|collection date:2024 06|geo loc name:USA: Maryland Bethesda|sex:n/a|tissue:Anterior to swimbladder|genotype:WT|replicate:wt 2 dpf replicate 1|BioSampleModel:Model organism or animal,,,,,,,,,RNA Seq of zebrafish TS2 mutant,wt1 d2,wt1 d2,TruSeq library,,,RNA-Seq,TRANSCRIPTOMIC,PolyA,PAIRED,ILLUMINA,Illumina NovaSeq 6000,,SRP582714,,,y680_WT-1_S9_R1_001.fastq.gz y680_WT-1_S9_R2_001.fastq.gz,fastq fastq,6639999850.0,33169283.0,y680 WT 1 S9 R1 001.fastq.gz,0:100.08 1:100.10,A:1706100512;C:1614640504;G:1665440844;T:1651930739;N:1887251,100,100,,,1706100512,1614640504,1665440844,1651930739,1887251,SRX28646802,SRS24917411,SRA2122396,NICHD|DDB,NICHD,,,,,,,,,,,,B,B,biological fallback assumption,illumina,novaseq_era,unknown,poly_a,trueseq,bulk,unknown,unknown,,United States,2025-05-01,Hatching,Embryo,Undetermined,Embryo Imprecise
38119,SRR1551796,SRX681416,SRS685079,SRP045504,PRJNA258223,Danio rerio strain:TL Transcriptome or Gene expression,PRJNA258223,Other,Transcriptome profiling of keratocytes derived from 2dpf and 4dpf embryos,,,,Transcriptome from 2dpf keratocytes,keratocytes from 2dpf embryos,,breed:TL|strain:TL|age:2dpf|biomaterial provider:Julie Theriot Stanford University|sex:not collected|tissue:keratocytes skin|cell type:keratocyte|dev stage:2dpf|BioSampleModel:Model organism or animal,,,,,,,,,Transcriptome from 2dpf keratocytes replicate 3,2dpf 3,2dpf 3,RNA was extracted from approximately 1 x 105 keratocytes per sample using Trizol Invitrogen. Three biological replicates were collected for each developmental stage. Ribosomal RNA was depleted using the Ribo Zero magnetic kit Epicentre. The remaining mRNA was then fragmented with 8 minutes incubation in 50mM sodium carbonate/bicarbonate 1mM EDTA pH 9.2 at 95 degrees. To prepare cDNA first strand synthesis was performed with Superscript III Invitrogen using random hexamer priming and second strand synthesis was performed with DNA Polymerase I NEB. Illumina libraries were prepared from the cDNA in an automated fashion using the Illumina TruSeq sample prep kit with TruSeq adapters on a SPRIworks System I Beckman Coulter. Sequencing reactions were performed on an Illumina Genome Analyzer IIX according to manufacturer’s instructions to generate 40nt single ended reads.,,,RNA-Seq,TRANSCRIPTOMIC,PolyA,SINGLE,ILLUMINA,Illumina Genome Analyzer IIx,400Application ReadForward1,SRP045504,,,lane3_2dpf629.fastq,fastq,551787520.0,13794688.0,2dpf 3,0:40,A:138660463;C:132605577;G:134640312;T:145852714;N:28454,40,,,,138660463,132605577,134640312,145852714,28454,SRX681416,SRS685079,SRA179323,Stanford University|Julie Theriot,Stanford University,1,0.91375,,0.21598,,0.75317,,0.47881,,40,,B,,usable mapping rate,illumina,early_illumina,unknown,poly_a,trueseq,bulk,unknown,unknown,,United States,2014-08-14,Hatching,Embryo,Skin,Surface Structure
38120,SRR1551795,SRX681415,SRS685079,SRP045504,PRJNA258223,Danio rerio strain:TL Transcriptome or Gene expression,PRJNA258223,Other,Transcriptome profiling of keratocytes derived from 2dpf and 4dpf embryos,,,,Transcriptome from 2dpf keratocytes,keratocytes from 2dpf embryos,,breed:TL|strain:TL|age:2dpf|biomaterial provider:Julie Theriot Stanford University|sex:not collected|tissue:keratocytes skin|cell type:keratocyte|dev stage:2dpf|BioSampleModel:Model organism or animal,,,,,,,,,Transcriptome from 2dpf keratocytes,2dpf 2,2dpf 2,RNA was extracted from approximately 1 x 105 keratocytes per sample using Trizol Invitrogen. Three biological replicates were collected for each developmental stage. Ribosomal RNA was depleted using the Ribo Zero magnetic kit Epicentre. The remaining mRNA was then fragmented with 8 minutes incubation in 50mM sodium carbonate/bicarbonate 1mM EDTA pH 9.2 at 95 degrees. To prepare cDNA first strand synthesis was performed with Superscript III Invitrogen using random hexamer priming and second strand synthesis was performed with DNA Polymerase I NEB. Illumina libraries were prepared from the cDNA in an automated fashion using the Illumina TruSeq sample prep kit with TruSeq adapters on a SPRIworks System I Beckman Coulter. Sequencing reactions were performed on an Illumina Genome Analyzer IIX according to manufacturer’s instructions to generate 40nt single ended reads.,,,RNA-Seq,TRANSCRIPTOMIC,PolyA,SINGLE,ILLUMINA,Illumina Genome Analyzer IIx,400Application ReadForward1,SRP045504,,,lane1_2dpf.fastq,fastq,796874112.0,22135392.0,2dpf 2,0:36,A:209956517;C:184659684;G:185142987;T:216055902;N:1059022,36,,,,209956517,184659684,185142987,216055902,1059022,SRX681415,SRS685079,SRA179323,Stanford University|Julie Theriot,Stanford University,1,0.89178,,0.17837,,0.7609,,0.49774,,36,,B,,usable mapping rate,illumina,early_illumina,unknown,poly_a,trueseq,bulk,unknown,unknown,,United States,2015-04-07,Hatching,Embryo,Skin,Surface Structure
38121,SRR1551782,SRX681402,SRS685079,SRP045504,PRJNA258223,Danio rerio strain:TL Transcriptome or Gene expression,PRJNA258223,Other,Transcriptome profiling of keratocytes derived from 2dpf and 4dpf embryos,,,,Transcriptome from 2dpf keratocytes,keratocytes from 2dpf embryos,,breed:TL|strain:TL|age:2dpf|biomaterial provider:Julie Theriot Stanford University|sex:not collected|tissue:keratocytes skin|cell type:keratocyte|dev stage:2dpf|BioSampleModel:Model organism or animal,,,,,,,,,2dpf keratocytes replicate 1,2dpf 1,2dpf 1,RNA was extracted from approximately 1 x 105 keratocytes per sample using Trizol Invitrogen. Three biological replicates were collected for each developmental stage. Ribosomal RNA was depleted using the Ribo Zero magnetic kit Epicentre. The remaining mRNA was then fragmented with 8 minutes incubation in 50mM sodium carbonate/bicarbonate 1mM EDTA pH 9.2 at 95 degrees. To prepare cDNA first strand synthesis was performed with Superscript III Invitrogen using random hexamer priming and second strand synthesis was performed with DNA Polymerase I NEB. Illumina libraries were prepared from the cDNA in an automated fashion using the Illumina TruSeq sample prep kit with TruSeq adapters on a SPRIworks System I Beckman Coulter. Sequencing reactions were performed on an Illumina Genome Analyzer IIX according to manufacturer’s instructions to generate 40nt single ended reads.,,,RNA-Seq,TRANSCRIPTOMIC,PolyA,SINGLE,ILLUMINA,Illumina Genome Analyzer IIx,400Application ReadForward1,SRP045504,,,lane6_2dpf.fastq,fastq,647944118.0,17051161.0,2dpf 1,0:38,A:145735255;C:167786338;G:186677806;T:141598169;N:6146550,38,,,,145735255,167786338,186677806,141598169,6146550,SRX681402,SRS685079,SRA179323,Stanford University|Julie Theriot,Stanford University,1,0.7695,,0.15816,,0.81491,,0.44972,,38,,B,,usable mapping rate,illumina,early_illumina,unknown,poly_a,trueseq,bulk,unknown,unknown,,United States,2014-08-14,Hatching,Embryo,Skin,Surface Structure
55877,SRR10863019,SRX7533050,SRS5972252,SRP241074,PRJNA597223,Danio rerio primordial germ cell expression pofiling,PRJNA597223,Whole Genome Sequencing,Primordial germ cells PGCs are the precursors of germ cells which migrate to the genital ridge during early development. Relatively little is known about PGCs post their migration. We studied this post migratory stage using microscopy and sequencing techniques. We found that many PGC specific genes including genes known to induce PGC fate in the mouse are only activated several days post migration. At this same timepoint PGC nuclei become extremely gyrated displaying general opening of chromatin and high levels of transcription. This is accompanied by changes in nuage morphology and expression of large loci named PERLs enriched for retro transposons and piRNAs. piRNA biogenesis signatures rise in this same period. Interestingly no nuclear Piwi protein could be detected indicating that the zebrafish piRNA pathway is fully cytoplasmic. Our data show that the post migratory stage of zebrafish PGCs holds many cues to both germ cell fate establishment and piRNA pathway activation.,,,The vasa:eGFP line Krøvel and Olsen 2002 was used to FACS sort PGCs at different timepoints. Embryos were collected and incubated with TrypLETM Express for 40 to 70 minutes killed on ice and gently pippeted up and down with a glass pipet and/or a 200µl low retention pipet tip. post visual inspection cell suspension was separated from trunks using a 100 µm siev. Following another 5 15 minutes of digestion FCS was added to 10% of total volume. Cells were spun down at 500g for 5min at RT washed with PBS resuspended in PBS with 2% FCS put on Ice and immediately subjected to FACS using a 85µm nozzle on a BD FACSAria III SORP Becton Dickinson. 1000 to 2500 cells were sorted directly into Trizol. RNA from sorted PGCs and whole embryos was extracted with Trizol and stored in MQ at 80°C until library preparation was done.,,wt PGCs 2dpf rep3,,strain:vasa:eGFP|isolate:not applicable|breed:TUE|cultivar:not applicable|ecotype:not applicable|age:2dpf|dev stage:Long pec stage|sex:not applicable|tissue:germline|biological replicate:replicate 3|BioSampleModel:Model organism or animal,,,,,,,,,mRNA of Zebrafish: PGCs 2dpf,imb ketting 2018 02 redl SmartSeq 06 61 2dpf PGCs rep3 S6,imb ketting 2018 02 redl SmartSeq 06 61 2dpf PGCs rep3 S6,Library prep was performed with SmartSeq2 RNA Seq System following NuGen`s standard protocol M01406v2. Libraries were prepared with a starting amount of 1 ng and amplified in 12 PCR cycles.,,,RNA-Seq,TRANSCRIPTOMIC,PolyA,PAIRED,ILLUMINA,NextSeq 550,,SRP241074,,,imb_ketting_2018_02_redl_SmartSeq_06_61_2dpf_PGCs_rep3_S6.R1.fastq.gz imb_ketting_2018_02_redl_SmartSeq_06_61_2dpf_PGCs_rep3_S6.R2.fastq.gz,fastq fastq,3108720942.0,19675449.0,imb ketting 2018 02 redl SmartSeq 06 61 2dpf PGCs rep3 S6.R1.fastq.gz,0:79 1:79,A:837919918;C:714812252;G:702998193;T:852776390;N:214189,79,79,,,837919918,714812252,702998193,852776390,214189,SRX7533050,SRS5972252,SRA1023320,Rene Ketting group|Ketting Lab,Rene Ketting group,2,0.93952,0.93945,0.07277,0.07278,0.66983,0.67221,0.50192,0.50133,79,79,B,B,biological fallback assumption,illumina,nextseq,unknown,poly_a,unknown,sc,single_cell_plate,smartseq,,Germany,2020-01-10,Hatching,Embryo,Whole Organism,All anatomical structures
55878,SRR10863020,SRX7533049,SRS5972244,SRP241074,PRJNA597223,Danio rerio primordial germ cell expression pofiling,PRJNA597223,Whole Genome Sequencing,Primordial germ cells PGCs are the precursors of germ cells which migrate to the genital ridge during early development. Relatively little is known about PGCs post their migration. We studied this post migratory stage using microscopy and sequencing techniques. We found that many PGC specific genes including genes known to induce PGC fate in the mouse are only activated several days post migration. At this same timepoint PGC nuclei become extremely gyrated displaying general opening of chromatin and high levels of transcription. This is accompanied by changes in nuage morphology and expression of large loci named PERLs enriched for retro transposons and piRNAs. piRNA biogenesis signatures rise in this same period. Interestingly no nuclear Piwi protein could be detected indicating that the zebrafish piRNA pathway is fully cytoplasmic. Our data show that the post migratory stage of zebrafish PGCs holds many cues to both germ cell fate establishment and piRNA pathway activation.,,,The vasa:eGFP line Krøvel and Olsen 2002 was used to FACS sort PGCs at different timepoints. Embryos were collected and incubated with TrypLETM Express for 40 to 70 minutes killed on ice and gently pippeted up and down with a glass pipet and/or a 200µl low retention pipet tip. post visual inspection cell suspension was separated from trunks using a 100 µm siev. Following another 5 15 minutes of digestion FCS was added to 10% of total volume. Cells were spun down at 500g for 5min at RT washed with PBS resuspended in PBS with 2% FCS put on Ice and immediately subjected to FACS using a 85µm nozzle on a BD FACSAria III SORP Becton Dickinson. 1000 to 2500 cells were sorted directly into Trizol. RNA from sorted PGCs and whole embryos was extracted with Trizol and stored in MQ at 80°C until library preparation was done.,,wt PGCs 2dpf rep2,,strain:vasa:eGFP|isolate:not applicable|breed:TUE|cultivar:not applicable|ecotype:not applicable|age:2dpf|dev stage:Long pec stage|sex:not applicable|tissue:germline|biological replicate:replicate 2|BioSampleModel:Model organism or animal,,,,,,,,,mRNA of Zebrafish: PGCs 2dpf,imb ketting 2018 02 redl SmartSeq 05 22 2dpf PGCs rep2 S5,imb ketting 2018 02 redl SmartSeq 05 22 2dpf PGCs rep2 S5,Library prep was performed with SmartSeq2 RNA Seq System following NuGen`s standard protocol M01406v2. Libraries were prepared with a starting amount of 1 ng and amplified in 12 PCR cycles.,,,RNA-Seq,TRANSCRIPTOMIC,PolyA,PAIRED,ILLUMINA,NextSeq 550,,SRP241074,,,imb_ketting_2018_02_redl_SmartSeq_05_22_2dpf_PGCs_rep2_S5.R1.fastq.gz imb_ketting_2018_02_redl_SmartSeq_05_22_2dpf_PGCs_rep2_S5.R2.fastq.gz,fastq fastq,3241530370.0,20516015.0,imb ketting 2018 02 redl SmartSeq 05 22 2dpf PGCs rep2 S5.R1.fastq.gz,0:79 1:79,A:861911078;C:762577918;G:730686512;T:886132359;N:222503,79,79,,,861911078,762577918,730686512,886132359,222503,SRX7533049,SRS5972244,SRA1023320,Rene Ketting group|Ketting Lab,Rene Ketting group,2,0.93797,0.93734,0.06416,0.06415,0.67004,0.67251,0.50367,0.50353,79,79,B,B,biological fallback assumption,illumina,nextseq,unknown,poly_a,unknown,sc,single_cell_plate,smartseq,,Germany,2020-01-10,Hatching,Embryo,Whole Organism,All anatomical structures
55879,SRR10863021,SRX7533048,SRS5972269,SRP241074,PRJNA597223,Danio rerio primordial germ cell expression pofiling,PRJNA597223,Whole Genome Sequencing,Primordial germ cells PGCs are the precursors of germ cells which migrate to the genital ridge during early development. Relatively little is known about PGCs post their migration. We studied this post migratory stage using microscopy and sequencing techniques. We found that many PGC specific genes including genes known to induce PGC fate in the mouse are only activated several days post migration. At this same timepoint PGC nuclei become extremely gyrated displaying general opening of chromatin and high levels of transcription. This is accompanied by changes in nuage morphology and expression of large loci named PERLs enriched for retro transposons and piRNAs. piRNA biogenesis signatures rise in this same period. Interestingly no nuclear Piwi protein could be detected indicating that the zebrafish piRNA pathway is fully cytoplasmic. Our data show that the post migratory stage of zebrafish PGCs holds many cues to both germ cell fate establishment and piRNA pathway activation.,,,The vasa:eGFP line Krøvel and Olsen 2002 was used to FACS sort PGCs at different timepoints. Embryos were collected and incubated with TrypLETM Express for 40 to 70 minutes killed on ice and gently pippeted up and down with a glass pipet and/or a 200µl low retention pipet tip. post visual inspection cell suspension was separated from trunks using a 100 µm siev. Following another 5 15 minutes of digestion FCS was added to 10% of total volume. Cells were spun down at 500g for 5min at RT washed with PBS resuspended in PBS with 2% FCS put on Ice and immediately subjected to FACS using a 85µm nozzle on a BD FACSAria III SORP Becton Dickinson. 1000 to 2500 cells were sorted directly into Trizol. RNA from sorted PGCs and whole embryos was extracted with Trizol and stored in MQ at 80°C until library preparation was done.,,wt PGCs 2dpf rep1,,strain:vasa:eGFP|isolate:not applicable|breed:TUE|cultivar:not applicable|ecotype:not applicable|age:2dpf|dev stage:Long pec stage|sex:not applicable|tissue:germline|biological replicate:replicate 1|BioSampleModel:Model organism or animal,,,,,,,,,mRNA of Zebrafish: PGCs 2dpf,imb ketting 2018 02 redl SmartSeq 04 07 2dpf PGCs rep1 S4,imb ketting 2018 02 redl SmartSeq 04 07 2dpf PGCs rep1 S4,Library prep was performed with SmartSeq2 RNA Seq System following NuGen`s standard protocol M01406v2. Libraries were prepared with a starting amount of 1 ng and amplified in 12 PCR cycles.,,,RNA-Seq,TRANSCRIPTOMIC,PolyA,PAIRED,ILLUMINA,NextSeq 550,,SRP241074,,,imb_ketting_2018_02_redl_SmartSeq_04_07_2dpf_PGCs_rep1_S4.R1.fastq.gz imb_ketting_2018_02_redl_SmartSeq_04_07_2dpf_PGCs_rep1_S4.R2.fastq.gz,fastq fastq,3363342524.0,21286978.0,imb ketting 2018 02 redl SmartSeq 04 07 2dpf PGCs rep1 S4.R1.fastq.gz,0:79 1:79,A:906938869;C:772810568;G:756589497;T:926768184;N:235406,79,79,,,906938869,772810568,756589497,926768184,235406,SRX7533048,SRS5972269,SRA1023320,Rene Ketting group|Ketting Lab,Rene Ketting group,2,0.93588,0.93667,0.11457,0.11483,0.66663,0.66799,0.4795,0.4762,79,79,B,B,biological fallback assumption,illumina,nextseq,unknown,poly_a,unknown,sc,single_cell_plate,smartseq,,Germany,2020-01-10,Hatching,Embryo,Whole Organism,All anatomical structures
55893,SRR10863034,SRX7533034,SRS5972259,SRP241074,PRJNA597223,Danio rerio primordial germ cell expression pofiling,PRJNA597223,Whole Genome Sequencing,Primordial germ cells PGCs are the precursors of germ cells which migrate to the genital ridge during early development. Relatively little is known about PGCs post their migration. We studied this post migratory stage using microscopy and sequencing techniques. We found that many PGC specific genes including genes known to induce PGC fate in the mouse are only activated several days post migration. At this same timepoint PGC nuclei become extremely gyrated displaying general opening of chromatin and high levels of transcription. This is accompanied by changes in nuage morphology and expression of large loci named PERLs enriched for retro transposons and piRNAs. piRNA biogenesis signatures rise in this same period. Interestingly no nuclear Piwi protein could be detected indicating that the zebrafish piRNA pathway is fully cytoplasmic. Our data show that the post migratory stage of zebrafish PGCs holds many cues to both germ cell fate establishment and piRNA pathway activation.,,,The vasa:eGFP line Krøvel and Olsen 2002 was used to FACS sort PGCs at different timepoints. Embryos were collected and incubated with TrypLETM Express for 40 to 70 minutes killed on ice and gently pippeted up and down with a glass pipet and/or a 200µl low retention pipet tip. post visual inspection cell suspension was separated from trunks using a 100 µm siev. Following another 5 15 minutes of digestion FCS was added to 10% of total volume. Cells were spun down at 500g for 5min at RT washed with PBS resuspended in PBS with 2% FCS put on Ice and immediately subjected to FACS using a 85µm nozzle on a BD FACSAria III SORP Becton Dickinson. 1000 to 2500 cells were sorted directly into Trizol. RNA from sorted PGCs and whole embryos was extracted with Trizol and stored in MQ at 80°C until library preparation was done.,,wt Fish 2dpf rep2,,strain:vasa:eGFP|isolate:not applicable|breed:TUE|cultivar:not applicable|ecotype:not applicable|age:2dpf|dev stage:Long pec stage|sex:not applicable|tissue:whole embryo|biological replicate:replicate 2|BioSampleModel:Model organism or animal,,,,,,,,,mRNA of Zebrafish: Fish 2dpf,imb ketting 2018 02 redl SmartSeq 16 63 2dpf Fish rep2 S16,imb ketting 2018 02 redl SmartSeq 16 63 2dpf Fish rep2 S16,Library prep was performed with SmartSeq2 RNA Seq System following NuGen`s standard protocol M01406v2. Libraries were prepared with a starting amount of 1 ng and amplified in 12 PCR cycles.,,,RNA-Seq,TRANSCRIPTOMIC,PolyA,PAIRED,ILLUMINA,NextSeq 550,,SRP241074,,,imb_ketting_2018_02_redl_SmartSeq_16_63_2dpf_Fish_rep2_S16.R1.fastq.gz imb_ketting_2018_02_redl_SmartSeq_16_63_2dpf_Fish_rep2_S16.R2.fastq.gz,fastq fastq,3091658206.0,19567457.0,imb ketting 2018 02 redl SmartSeq 16 63 2dpf Fish rep2 S16.R1.fastq.gz,0:79 1:79,A:818837054;C:727836511;G:707557580;T:837216941;N:210120,79,79,,,818837054,727836511,707557580,837216941,210120,SRX7533034,SRS5972259,SRA1023320,Rene Ketting group|Ketting Lab,Rene Ketting group,2,0.94667,0.94646,0.08702,0.08692,0.69804,0.70138,0.43628,0.43409,79,79,B,B,biological fallback assumption,illumina,nextseq,unknown,poly_a,unknown,sc,single_cell_plate,smartseq,,Germany,2020-01-10,Hatching,Embryo,Whole Organism,All anatomical structures
55894,SRR10863035,SRX7533033,SRS5972258,SRP241074,PRJNA597223,Danio rerio primordial germ cell expression pofiling,PRJNA597223,Whole Genome Sequencing,Primordial germ cells PGCs are the precursors of germ cells which migrate to the genital ridge during early development. Relatively little is known about PGCs post their migration. We studied this post migratory stage using microscopy and sequencing techniques. We found that many PGC specific genes including genes known to induce PGC fate in the mouse are only activated several days post migration. At this same timepoint PGC nuclei become extremely gyrated displaying general opening of chromatin and high levels of transcription. This is accompanied by changes in nuage morphology and expression of large loci named PERLs enriched for retro transposons and piRNAs. piRNA biogenesis signatures rise in this same period. Interestingly no nuclear Piwi protein could be detected indicating that the zebrafish piRNA pathway is fully cytoplasmic. Our data show that the post migratory stage of zebrafish PGCs holds many cues to both germ cell fate establishment and piRNA pathway activation.,,,The vasa:eGFP line Krøvel and Olsen 2002 was used to FACS sort PGCs at different timepoints. Embryos were collected and incubated with TrypLETM Express for 40 to 70 minutes killed on ice and gently pippeted up and down with a glass pipet and/or a 200µl low retention pipet tip. post visual inspection cell suspension was separated from trunks using a 100 µm siev. Following another 5 15 minutes of digestion FCS was added to 10% of total volume. Cells were spun down at 500g for 5min at RT washed with PBS resuspended in PBS with 2% FCS put on Ice and immediately subjected to FACS using a 85µm nozzle on a BD FACSAria III SORP Becton Dickinson. 1000 to 2500 cells were sorted directly into Trizol. RNA from sorted PGCs and whole embryos was extracted with Trizol and stored in MQ at 80°C until library preparation was done.,,wt Fish 2dpf rep1,,strain:vasa:eGFP|isolate:not applicable|breed:TUE|cultivar:not applicable|ecotype:not applicable|age:2dpf|dev stage:Long pec stage|sex:not applicable|tissue:whole embryo|biological replicate:replicate 1|BioSampleModel:Model organism or animal,,,,,,,,,mRNA of Zebrafish: Fish 2dpf,imb ketting 2018 02 redl SmartSeq 15 02 2dpf Fish rep1 S15,imb ketting 2018 02 redl SmartSeq 15 02 2dpf Fish rep1 S15,Library prep was performed with SmartSeq2 RNA Seq System following NuGen`s standard protocol M01406v2. Libraries were prepared with a starting amount of 1 ng and amplified in 12 PCR cycles.,,,RNA-Seq,TRANSCRIPTOMIC,PolyA,PAIRED,ILLUMINA,NextSeq 550,,SRP241074,,,imb_ketting_2018_02_redl_SmartSeq_15_02_2dpf_Fish_rep1_S15.R1.fastq.gz imb_ketting_2018_02_redl_SmartSeq_15_02_2dpf_Fish_rep1_S15.R2.fastq.gz,fastq fastq,3499876328.0,22151116.0,imb ketting 2018 02 redl SmartSeq 15 02 2dpf Fish rep1 S15.R1.fastq.gz,0:79 1:79,A:931187812;C:820606968;G:796104654;T:951733888;N:243006,79,79,,,931187812,820606968,796104654,951733888,243006,SRX7533033,SRS5972258,SRA1023320,Rene Ketting group|Ketting Lab,Rene Ketting group,2,0.94532,0.94537,0.10394,0.10459,0.69489,0.69727,0.4345,0.43676,79,79,B,B,biological fallback assumption,illumina,nextseq,unknown,poly_a,unknown,sc,single_cell_plate,smartseq,,Germany,2020-01-10,Hatching,Embryo,Whole Organism,All anatomical structures
59036,SRR11578951,SRX8146805,SRS6509417,SRP257662,PRJNA627095,Transcriptional profile of SAG neurod+ neuroblasts from clo mutant and sibling embryos at 54 hpf,PRJNA627095,Other,In many organs stem cell function depends on the communication with their niche partners. Cranial sensory neurons develop in close proximity to blood vessels however whether vasculature is an integral component of their niches is yet unknown. Here two separate novel roles for vasculature in cranial sensory neurogenesis in zebrafish are uncovered. The first involves a restrain of neuroblast proliferation at early stages. The second instead requires blood flow to trigger a transcriptional response which has been identified thanks to a RNAseq analysis. This transcriptional change modifies neuroblast metabolic status and induces sensory neuron differentiation. Altogether we demonstrate that the cranial vasculature constitutes a hitherto unrecognized niche component of the sensory ganglia that regulates the pace of their growth and differentiation dynamics.,,,WT,54WT4,54WT4,,strain:TgBACneurod:gfpnl1|age:54 hpf|sex:not applicable|tissue:SAG tissue|collection date:2018 11 14|geo loc name:Spain: Barcelona|lat lon:41.3851 N 2.1734 E|filename:54WT4 R1 001.fastq.gz|filename2:54WT4 R2 001.fastq.gz|BioSampleModel:Model organism or animal,,,,,,,,,54WT4,S7,S7,low input mRNA sequencing,,,RNA-Seq,TRANSCRIPTOMIC,PolyA,PAIRED,ILLUMINA,NextSeq 500,,SRP257662,,,54WT4_R2_001.fastq.gz 54WT4_R1_001.fastq.gz,fastq fastq,7136583059.0,47328719.0,54WT4 R1 001.fastq.gz,0:75.39 1:75.40,A:1980254600;C:1593971374;G:1644375041;T:1916731613;N:1250431,75,75,,,1980254600,1593971374,1644375041,1916731613,1250431,SRX8146805,SRS6509417,SRA1067518,UPF|Genomics,UPF,2,0.91504,0.91439,0.09602,0.09527,0.73245,0.73777,0.46955,0.48655,76,76,B,B,biological fallback assumption,illumina,nextseq,unknown,poly_a,unknown,bulk,unknown,unknown,,Unknown,2020-04-21,Hatching,Embryo,Undetermined,Embryo Imprecise
59037,SRR11578952,SRX8146804,SRS6509416,SRP257662,PRJNA627095,Transcriptional profile of SAG neurod+ neuroblasts from clo mutant and sibling embryos at 54 hpf,PRJNA627095,Other,In many organs stem cell function depends on the communication with their niche partners. Cranial sensory neurons develop in close proximity to blood vessels however whether vasculature is an integral component of their niches is yet unknown. Here two separate novel roles for vasculature in cranial sensory neurogenesis in zebrafish are uncovered. The first involves a restrain of neuroblast proliferation at early stages. The second instead requires blood flow to trigger a transcriptional response which has been identified thanks to a RNAseq analysis. This transcriptional change modifies neuroblast metabolic status and induces sensory neuron differentiation. Altogether we demonstrate that the cranial vasculature constitutes a hitherto unrecognized niche component of the sensory ganglia that regulates the pace of their growth and differentiation dynamics.,,,WT,54WT3,54WT3,,strain:TgBACneurod:gfpnl1|age:54 hpf|sex:not applicable|tissue:SAG tissue|collection date:2018 11 14|geo loc name:Spain: Barcelona|lat lon:41.3851 N 2.1734 E|filename:54WT3 R1 001.fastq.gz|filename2:54WT3 R2 001.fastq.gz|BioSampleModel:Model organism or animal,,,,,,,,,54WT3,S5,S5,low input mRNA sequencing,,,RNA-Seq,TRANSCRIPTOMIC,PolyA,PAIRED,ILLUMINA,NextSeq 500,,SRP257662,,,54WT3_R1_001.fastq.gz 54WT3_R2_001.fastq.gz,fastq fastq,5683476624.0,37713746.0,54WT3 R1 001.fastq.gz,0:75.35 1:75.35,A:1578885174;C:1271523804;G:1309222456;T:1522827535;N:1017655,75,75,,,1578885174,1271523804,1309222456,1522827535,1017655,SRX8146804,SRS6509416,SRA1067518,UPF|Genomics,UPF,2,0.91126,0.91193,0.10356,0.10472,0.73975,0.7461,0.47619,0.47737,75,75,B,B,biological fallback assumption,illumina,nextseq,unknown,poly_a,unknown,bulk,unknown,unknown,,Unknown,2020-04-21,Hatching,Embryo,Undetermined,Embryo Imprecise
59038,SRR11578953,SRX8146803,SRS6509415,SRP257662,PRJNA627095,Transcriptional profile of SAG neurod+ neuroblasts from clo mutant and sibling embryos at 54 hpf,PRJNA627095,Other,In many organs stem cell function depends on the communication with their niche partners. Cranial sensory neurons develop in close proximity to blood vessels however whether vasculature is an integral component of their niches is yet unknown. Here two separate novel roles for vasculature in cranial sensory neurogenesis in zebrafish are uncovered. The first involves a restrain of neuroblast proliferation at early stages. The second instead requires blood flow to trigger a transcriptional response which has been identified thanks to a RNAseq analysis. This transcriptional change modifies neuroblast metabolic status and induces sensory neuron differentiation. Altogether we demonstrate that the cranial vasculature constitutes a hitherto unrecognized niche component of the sensory ganglia that regulates the pace of their growth and differentiation dynamics.,,,WT,54WT2,54WT2,,strain:TgBACneurod:gfpnl1|age:54 hpf|sex:not applicable|tissue:SAG tissue|collection date:2018 11 14|geo loc name:Spain: Barcelona|lat lon:41.3851 N 2.1734 E|filename:54WT2 R1 001.fastq.gz|filename2:54WT2 R2 001.fastq.gz|BioSampleModel:Model organism or animal,,,,,,,,,54WT2,S3,S3,low input mRNA sequencing,,,RNA-Seq,TRANSCRIPTOMIC,PolyA,PAIRED,ILLUMINA,NextSeq 500,,SRP257662,,,54WT2_R1_001.fastq.gz 54WT2_R2_001.fastq.gz,fastq fastq,6860136869.0,45523385.0,54WT2 R1 001.fastq.gz,0:75.34 1:75.35,A:1906640944;C:1530528609;G:1580010044;T:1841799217;N:1158055,75,75,,,1906640944,1530528609,1580010044,1841799217,1158055,SRX8146803,SRS6509415,SRA1067518,UPF|Genomics,UPF,2,0.91215,0.91207,0.10861,0.11028,0.74381,0.74968,0.48908,0.49262,76,76,B,B,biological fallback assumption,illumina,nextseq,unknown,poly_a,unknown,bulk,unknown,unknown,,Unknown,2020-04-21,Hatching,Embryo,Undetermined,Embryo Imprecise
59039,SRR11578954,SRX8146802,SRS6509414,SRP257662,PRJNA627095,Transcriptional profile of SAG neurod+ neuroblasts from clo mutant and sibling embryos at 54 hpf,PRJNA627095,Other,In many organs stem cell function depends on the communication with their niche partners. Cranial sensory neurons develop in close proximity to blood vessels however whether vasculature is an integral component of their niches is yet unknown. Here two separate novel roles for vasculature in cranial sensory neurogenesis in zebrafish are uncovered. The first involves a restrain of neuroblast proliferation at early stages. The second instead requires blood flow to trigger a transcriptional response which has been identified thanks to a RNAseq analysis. This transcriptional change modifies neuroblast metabolic status and induces sensory neuron differentiation. Altogether we demonstrate that the cranial vasculature constitutes a hitherto unrecognized niche component of the sensory ganglia that regulates the pace of their growth and differentiation dynamics.,,,WT,54WT1,54WT1,,strain:TgBACneurod:gfpnl1|age:54 hpf|sex:not applicable|tissue:SAG tissue|collection date:2018 11 14|geo loc name:Spain: Barcelona|lat lon:41.3851 N 2.1734 E|filename:54WT1 R1 001.fastq.gz|filename2:54WT1 R2 001.fastq.gz|BioSampleModel:Model organism or animal,,,,,,,,,54WT1,S1,S1,low input mRNA sequencing,,,RNA-Seq,TRANSCRIPTOMIC,PolyA,PAIRED,ILLUMINA,NextSeq 500,,SRP257662,,,54WT1_R1_001.fastq.gz 54WT1_R2_001.fastq.gz,fastq fastq,6225284142.0,41319897.0,54WT1 R1 001.fastq.gz,0:75.33 1:75.33,A:1729358086;C:1392766589;G:1430690093;T:1671396108;N:1073266,75,75,,,1729358086,1392766589,1430690093,1671396108,1073266,SRX8146802,SRS6509414,SRA1067518,UPF|Genomics,UPF,2,0.92733,0.92867,0.09387,0.09333,0.71163,0.71494,0.486,0.48867,72,76,B,B,biological fallback assumption,illumina,nextseq,unknown,poly_a,unknown,bulk,unknown,unknown,,Unknown,2020-04-21,Hatching,Embryo,Undetermined,Embryo Imprecise
59040,SRR11578955,SRX8146801,SRS6509413,SRP257662,PRJNA627095,Transcriptional profile of SAG neurod+ neuroblasts from clo mutant and sibling embryos at 54 hpf,PRJNA627095,Other,In many organs stem cell function depends on the communication with their niche partners. Cranial sensory neurons develop in close proximity to blood vessels however whether vasculature is an integral component of their niches is yet unknown. Here two separate novel roles for vasculature in cranial sensory neurogenesis in zebrafish are uncovered. The first involves a restrain of neuroblast proliferation at early stages. The second instead requires blood flow to trigger a transcriptional response which has been identified thanks to a RNAseq analysis. This transcriptional change modifies neuroblast metabolic status and induces sensory neuron differentiation. Altogether we demonstrate that the cranial vasculature constitutes a hitherto unrecognized niche component of the sensory ganglia that regulates the pace of their growth and differentiation dynamics.,,,Clo,54Clo4,54Clo4,,strain:TgBACneurod:gfpnl1|age:54 hpf|sex:not applicable|tissue:SAG tissue|collection date:2018 11 14|geo loc name:Spain: Barcelona|lat lon:41.3851 N 2.1734 E|filename:54Clo4 R1 001.fastq.gz|filename2:54Clo4 R2 001.fastq.gz|BioSampleModel:Model organism or animal,,,,,,,,,54Clo4,S8,S8,low input mRNA sequencing,,,RNA-Seq,TRANSCRIPTOMIC,PolyA,PAIRED,ILLUMINA,NextSeq 500,,SRP257662,,,54Clo4_R1_001.fastq.gz 54Clo4_R2_001.fastq.gz,fastq fastq,6770856878.0,44915858.0,54Clo4 R1 001.fastq.gz,0:75.37 1:75.38,A:1876646232;C:1514408795;G:1561029116;T:1817557290;N:1215445,75,75,,,1876646232,1514408795,1561029116,1817557290,1215445,SRX8146801,SRS6509413,SRA1067518,UPF|Genomics,UPF,2,0.91659,0.91674,0.10388,0.10458,0.71948,0.72523,0.48917,0.4856,76,74,B,B,biological fallback assumption,illumina,nextseq,unknown,poly_a,unknown,bulk,unknown,unknown,,Unknown,2020-04-21,Hatching,Embryo,Undetermined,Embryo Imprecise
59041,SRR11578956,SRX8146800,SRS6509412,SRP257662,PRJNA627095,Transcriptional profile of SAG neurod+ neuroblasts from clo mutant and sibling embryos at 54 hpf,PRJNA627095,Other,In many organs stem cell function depends on the communication with their niche partners. Cranial sensory neurons develop in close proximity to blood vessels however whether vasculature is an integral component of their niches is yet unknown. Here two separate novel roles for vasculature in cranial sensory neurogenesis in zebrafish are uncovered. The first involves a restrain of neuroblast proliferation at early stages. The second instead requires blood flow to trigger a transcriptional response which has been identified thanks to a RNAseq analysis. This transcriptional change modifies neuroblast metabolic status and induces sensory neuron differentiation. Altogether we demonstrate that the cranial vasculature constitutes a hitherto unrecognized niche component of the sensory ganglia that regulates the pace of their growth and differentiation dynamics.,,,Clo,54Clo3,54Clo3,,strain:TgBACneurod:gfpnl1|age:54 hpf|sex:not applicable|tissue:SAG tissue|collection date:2018 11 14|geo loc name:Spain: Barcelona|lat lon:41.3851 N 2.1734 E|filename:54Clo3 R1 001.fastq.gz|filename2:54Clo3 R2 001.fastq.gz|BioSampleModel:Model organism or animal,,,,,,,,,54Clo3,S6,S6,low input mRNA sequencing,,,RNA-Seq,TRANSCRIPTOMIC,PolyA,PAIRED,ILLUMINA,NextSeq 500,,SRP257662,,,54Clo3_R2_001.fastq.gz 54Clo3_R1_001.fastq.gz,fastq fastq,5822390786.0,38650567.0,54Clo3 R1 001.fastq.gz,0:75.32 1:75.32,A:1619353584;C:1299190067;G:1337336722;T:1565486844;N:1023569,75,75,,,1619353584,1299190067,1337336722,1565486844,1023569,SRX8146800,SRS6509412,SRA1067518,UPF|Genomics,UPF,2,0.91509,0.91425,0.10591,0.10604,0.76386,0.7693,0.46813,0.46524,76,76,B,B,biological fallback assumption,illumina,nextseq,unknown,poly_a,unknown,bulk,unknown,unknown,,Unknown,2020-04-21,Hatching,Embryo,Undetermined,Embryo Imprecise
59042,SRR11578957,SRX8146799,SRS6509411,SRP257662,PRJNA627095,Transcriptional profile of SAG neurod+ neuroblasts from clo mutant and sibling embryos at 54 hpf,PRJNA627095,Other,In many organs stem cell function depends on the communication with their niche partners. Cranial sensory neurons develop in close proximity to blood vessels however whether vasculature is an integral component of their niches is yet unknown. Here two separate novel roles for vasculature in cranial sensory neurogenesis in zebrafish are uncovered. The first involves a restrain of neuroblast proliferation at early stages. The second instead requires blood flow to trigger a transcriptional response which has been identified thanks to a RNAseq analysis. This transcriptional change modifies neuroblast metabolic status and induces sensory neuron differentiation. Altogether we demonstrate that the cranial vasculature constitutes a hitherto unrecognized niche component of the sensory ganglia that regulates the pace of their growth and differentiation dynamics.,,,Clo,54Clo2,54Clo2,,strain:TgBACneurod:gfpnl1|age:54 hpf|sex:not applicable|tissue:SAG tissue|collection date:2018 11 14|geo loc name:Spain: Barcelona|lat lon:41.3851 N 2.1734 E|filename:54Clo2 R1 001.fastq.gz|filename2:54Clo2 R2 001.fastq.gz|BioSampleModel:Model organism or animal,,,,,,,,,54Clo2,S4,S4,low input mRNA sequencing,,,RNA-Seq,TRANSCRIPTOMIC,PolyA,PAIRED,ILLUMINA,NextSeq 500,,SRP257662,,,54Clo2_R2_001.fastq.gz 54Clo2_R1_001.fastq.gz,fastq fastq,6138626977.0,40727824.0,54Clo2 R1 001.fastq.gz,0:75.36 1:75.36,A:1700144556;C:1378599434;G:1420945150;T:1637894336;N:1043501,75,75,,,1700144556,1378599434,1420945150,1637894336,1043501,SRX8146799,SRS6509411,SRA1067518,UPF|Genomics,UPF,2,0.90602,0.90662,0.09596,0.09691,0.75872,0.76274,0.48174,0.48196,75,75,B,B,biological fallback assumption,illumina,nextseq,unknown,poly_a,unknown,bulk,unknown,unknown,,Unknown,2020-04-21,Hatching,Embryo,Undetermined,Embryo Imprecise
59043,SRR11578958,SRX8146798,SRS6509410,SRP257662,PRJNA627095,Transcriptional profile of SAG neurod+ neuroblasts from clo mutant and sibling embryos at 54 hpf,PRJNA627095,Other,In many organs stem cell function depends on the communication with their niche partners. Cranial sensory neurons develop in close proximity to blood vessels however whether vasculature is an integral component of their niches is yet unknown. Here two separate novel roles for vasculature in cranial sensory neurogenesis in zebrafish are uncovered. The first involves a restrain of neuroblast proliferation at early stages. The second instead requires blood flow to trigger a transcriptional response which has been identified thanks to a RNAseq analysis. This transcriptional change modifies neuroblast metabolic status and induces sensory neuron differentiation. Altogether we demonstrate that the cranial vasculature constitutes a hitherto unrecognized niche component of the sensory ganglia that regulates the pace of their growth and differentiation dynamics.,,,Clo,54Clo1,54Clo1,,strain:TgBACneurod:gfpnl1|age:54 hpf|sex:not applicable|tissue:SAG tissue|collection date:2018 11 14|geo loc name:Spain: Barcelona|lat lon:41.3851 N 2.1734 E|filename:54Clo1 R1 001.fastq.gz|filename2:54Clo1 R2 001.fastq.gz|BioSampleModel:Model organism or animal,,,,,,,,,54Clo1,S2,S2,low input mRNA sequencing,,,RNA-Seq,TRANSCRIPTOMIC,PolyA,PAIRED,ILLUMINA,NextSeq 500,,SRP257662,,,54Clo1_R1_001.fastq.gz 54Clo1_R2_001.fastq.gz,fastq fastq,7414571165.0,49175642.0,54Clo1 R1 001.fastq.gz,0:75.39 1:75.39,A:2056892212;C:1660481957;G:1708780731;T:1987161426;N:1254839,75,75,,,2056892212,1660481957,1708780731,1987161426,1254839,SRX8146798,SRS6509410,SRA1067518,UPF|Genomics,UPF,2,0.90938,0.91029,0.1024,0.1024,0.73507,0.73852,0.48964,0.48917,76,76,B,B,biological fallback assumption,illumina,nextseq,unknown,poly_a,unknown,bulk,unknown,unknown,,Unknown,2020-04-21,Hatching,Embryo,Undetermined,Embryo Imprecise