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682 rows where experiment.library_selection = "other", experiment.library_source = "TRANSCRIPTOMIC" and experiment.library_strategy = "OTHER"

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tissue_curation >10

devstage_curation >10

technology 7

devstage_curation_coarse 4

experiment.platform 3

experiment.library_layout 2

experiment.library_strategy 1

  • OTHER · 682 ✖

experiment.library_source 1

  • TRANSCRIPTOMIC · 682 ✖

experiment.library_selection 1

  • other · 682 ✖
Link 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
9717 9717 ERR3842002 ERX3854564 ERS4268611 ERP116106 PRJEB33323 Deconstructing the individual steps of vertebrate translation initiation ena-STUDY-Computational Biology Unit-03-07-2019-10:11:34:314-422 Other In eukaryotes the number of ribosomes synthesizing a given protein depends on how many are recruited to its mRNA their success in navigating its five prime untranslated region UTR and whether they recognize its start codon. Initiation of translation is a rate limiting step in protein synthesis and key to gene expression control 1 but despite this centrality it remains poorly understood 2 3. Here we introduce ribosome complex profiling RCP seq to capture the transcriptome wide occupancy of scanning initiating elongating and terminating ribosome complexes in a higher eukaryote. We track scanning and elongating ribosomes across all five prime UTRs in zebrafish which enable us to assess the individual regulatory contributions from the three stages of initiation: ribosome recruitment scanning of the five prime UTR and recognition of the start codon. Our data sheds light on small subunit recruitment to mRNAs presenting evidence for the threading model and demonstrates that sequence features regulate this recruitment. We estimate the processivity of scanning ribosomes as they traverse the five prime UTR and show that the repressive effects of upstream open reading frames depend on the efficiency of both translation initiation and termination. Finally we determine the optimal initiation contexts by directly estimating the conversion of scanning to elongating ribosomes and demonstrate specific regulation of translation initiation at the endoplasmic reticulum. Our results open for the possibility of deconvoluting translation initiation into separate stages and provides the first view of global occupancy of ribosomal small subunits in a vertebrate. ENA FIRST PUBLIC:2020 03 27|ENA LAST UPDATE:2019 07 03     Shield 4Ei SAMEA6504165 Computational Biology Unit ENA FIRST PUBLIC:2020 03 27T17:05:06Z|ENA LAST UPDATE:2020 01 27T16:04:35Z|External Id:SAMEA6504165|INSDC center name:Computational Biology Unit|INSDC first public:2020 03 27T17:05:06Z|INSDC last update:2020 01 27T16:04:35Z|INSDC status:public|Submitter Id:Shield 4Ei|common name:zebrafish|sample name:Shield 4Ei|scientific name:Danio rerio                 NextSeq 500 sequencing ena EXPERIMENT Computational Biology Unit 27 01 2020 17:26:02:557 2 Shield 4Ei LSU OTHER RCP seq   OTHER TRANSCRIPTOMIC other SINGLE ILLUMINA NextSeq 500   ERP116106 NextSeq 500 sequencing ENA FIRST PUBLIC:2020 03 27|ENA LAST UPDATE:2020 02 14     10915827408.0 143629308.0 ena RUN Computational Biology Unit 27 01 2020 17:26:02:557 2 0:76 A:3900515347;C:2409375725;G:3041696977;T:1564127293;N:112066 76       3900515347 2409375725 3041696977 1564127293 112066 ERX3854564 ERS4268611 ERA2359340 Computational Biology Unit|European Nucleotide Archive Computational Biology Unit 1 0.64398   0.40944   0.98817   0.59337   76   B   usable mapping rate illumina nextseq unknown other unknown bulk unknown unknown   Unknown 2019-07-03 Gastrula Embryo Undetermined Embryo Imprecise
9718 9718 ERR3842001 ERX3854563 ERS4268611 ERP116106 PRJEB33323 Deconstructing the individual steps of vertebrate translation initiation ena-STUDY-Computational Biology Unit-03-07-2019-10:11:34:314-422 Other In eukaryotes the number of ribosomes synthesizing a given protein depends on how many are recruited to its mRNA their success in navigating its five prime untranslated region UTR and whether they recognize its start codon. Initiation of translation is a rate limiting step in protein synthesis and key to gene expression control 1 but despite this centrality it remains poorly understood 2 3. Here we introduce ribosome complex profiling RCP seq to capture the transcriptome wide occupancy of scanning initiating elongating and terminating ribosome complexes in a higher eukaryote. We track scanning and elongating ribosomes across all five prime UTRs in zebrafish which enable us to assess the individual regulatory contributions from the three stages of initiation: ribosome recruitment scanning of the five prime UTR and recognition of the start codon. Our data sheds light on small subunit recruitment to mRNAs presenting evidence for the threading model and demonstrates that sequence features regulate this recruitment. We estimate the processivity of scanning ribosomes as they traverse the five prime UTR and show that the repressive effects of upstream open reading frames depend on the efficiency of both translation initiation and termination. Finally we determine the optimal initiation contexts by directly estimating the conversion of scanning to elongating ribosomes and demonstrate specific regulation of translation initiation at the endoplasmic reticulum. Our results open for the possibility of deconvoluting translation initiation into separate stages and provides the first view of global occupancy of ribosomal small subunits in a vertebrate. ENA FIRST PUBLIC:2020 03 27|ENA LAST UPDATE:2019 07 03     Shield 4Ei SAMEA6504165 Computational Biology Unit ENA FIRST PUBLIC:2020 03 27T17:05:06Z|ENA LAST UPDATE:2020 01 27T16:04:35Z|External Id:SAMEA6504165|INSDC center name:Computational Biology Unit|INSDC first public:2020 03 27T17:05:06Z|INSDC last update:2020 01 27T16:04:35Z|INSDC status:public|Submitter Id:Shield 4Ei|common name:zebrafish|sample name:Shield 4Ei|scientific name:Danio rerio                 NextSeq 500 sequencing ena EXPERIMENT Computational Biology Unit 27 01 2020 17:26:02:557 1 Shield 4Ei SSU OTHER RCP seq   OTHER TRANSCRIPTOMIC other SINGLE ILLUMINA NextSeq 500   ERP116106 NextSeq 500 sequencing ENA FIRST PUBLIC:2020 03 27|ENA LAST UPDATE:2020 02 14     7154041880.0 94132130.0 ena RUN Computational Biology Unit 27 01 2020 17:26:02:557 1 0:76 A:2939474250;C:1489083073;G:1922522149;T:802890185;N:72223 76       2939474250 1489083073 1922522149 802890185 72223 ERX3854563 ERS4268611 ERA2359340 Computational Biology Unit|European Nucleotide Archive Computational Biology Unit 1 0.4369   0.25417   0.9867   0.60047   76   B   usable mapping rate illumina nextseq unknown other unknown bulk unknown unknown   Unknown 2019-07-03 Gastrula Embryo Undetermined Embryo Imprecise
9729 9729 ERR3489881 ERX3511296 ERS3556004 ERP116106 PRJEB33323 Deconstructing the individual steps of vertebrate translation initiation ena-STUDY-Computational Biology Unit-03-07-2019-10:11:34:314-422 Other In eukaryotes the number of ribosomes synthesizing a given protein depends on how many are recruited to its mRNA their success in navigating its five prime untranslated region UTR and whether they recognize its start codon. Initiation of translation is a rate limiting step in protein synthesis and key to gene expression control 1 but despite this centrality it remains poorly understood 2 3. Here we introduce ribosome complex profiling RCP seq to capture the transcriptome wide occupancy of scanning initiating elongating and terminating ribosome complexes in a higher eukaryote. We track scanning and elongating ribosomes across all five prime UTRs in zebrafish which enable us to assess the individual regulatory contributions from the three stages of initiation: ribosome recruitment scanning of the five prime UTR and recognition of the start codon. Our data sheds light on small subunit recruitment to mRNAs presenting evidence for the threading model and demonstrates that sequence features regulate this recruitment. We estimate the processivity of scanning ribosomes as they traverse the five prime UTR and show that the repressive effects of upstream open reading frames depend on the efficiency of both translation initiation and termination. Finally we determine the optimal initiation contexts by directly estimating the conversion of scanning to elongating ribosomes and demonstrate specific regulation of translation initiation at the endoplasmic reticulum. Our results open for the possibility of deconvoluting translation initiation into separate stages and provides the first view of global occupancy of ribosomal small subunits in a vertebrate. ENA FIRST PUBLIC:2020 03 27|ENA LAST UPDATE:2019 07 03     Shield 1 SAMEA5752545 Computational Biology Unit ENA FIRST PUBLIC:2020 03 27T17:04:59Z|ENA LAST UPDATE:2019 07 03T10:01:14Z|External Id:SAMEA5752545|INSDC center name:Computational Biology Unit|INSDC first public:2020 03 27T17:04:59Z|INSDC last update:2019 07 03T10:01:14Z|INSDC status:public|Submitter Id:8|common name:zebrafish|dev stage:Shield|sample name:8|scientific name:Danio rerio                 NextSeq 500 sequencing ena EXPERIMENT Computational Biology Unit 22 08 2019 11:57:43:424 33 Shield 1 F20 None RCP seq   OTHER TRANSCRIPTOMIC other SINGLE ILLUMINA NextSeq 500   ERP116106 NextSeq 500 sequencing ENA FIRST PUBLIC:2020 03 27|ENA LAST UPDATE:2019 08 22     975578636.0 12836561.0 ena RUN Computational Biology Unit 22 08 2019 11:57:43:424 33 0:76 A:398325549;C:237563934;G:230721299;T:108957698;N:10156 76       398325549 237563934 230721299 108957698 10156 ERX3511296 ERS3556004 ERA2100634 Computational Biology Unit|European Nucleotide Archive Computational Biology Unit 1 0.33102   0.19766   0.99918   0.12812   76   B   usable mapping rate illumina nextseq unknown other unknown bulk unknown unknown   Unknown 2019-07-03 Gastrula Embryo Undetermined Embryo Imprecise
9730 9730 ERR3489880 ERX3511295 ERS3556004 ERP116106 PRJEB33323 Deconstructing the individual steps of vertebrate translation initiation ena-STUDY-Computational Biology Unit-03-07-2019-10:11:34:314-422 Other In eukaryotes the number of ribosomes synthesizing a given protein depends on how many are recruited to its mRNA their success in navigating its five prime untranslated region UTR and whether they recognize its start codon. Initiation of translation is a rate limiting step in protein synthesis and key to gene expression control 1 but despite this centrality it remains poorly understood 2 3. Here we introduce ribosome complex profiling RCP seq to capture the transcriptome wide occupancy of scanning initiating elongating and terminating ribosome complexes in a higher eukaryote. We track scanning and elongating ribosomes across all five prime UTRs in zebrafish which enable us to assess the individual regulatory contributions from the three stages of initiation: ribosome recruitment scanning of the five prime UTR and recognition of the start codon. Our data sheds light on small subunit recruitment to mRNAs presenting evidence for the threading model and demonstrates that sequence features regulate this recruitment. We estimate the processivity of scanning ribosomes as they traverse the five prime UTR and show that the repressive effects of upstream open reading frames depend on the efficiency of both translation initiation and termination. Finally we determine the optimal initiation contexts by directly estimating the conversion of scanning to elongating ribosomes and demonstrate specific regulation of translation initiation at the endoplasmic reticulum. Our results open for the possibility of deconvoluting translation initiation into separate stages and provides the first view of global occupancy of ribosomal small subunits in a vertebrate. ENA FIRST PUBLIC:2020 03 27|ENA LAST UPDATE:2019 07 03     Shield 1 SAMEA5752545 Computational Biology Unit ENA FIRST PUBLIC:2020 03 27T17:04:59Z|ENA LAST UPDATE:2019 07 03T10:01:14Z|External Id:SAMEA5752545|INSDC center name:Computational Biology Unit|INSDC first public:2020 03 27T17:04:59Z|INSDC last update:2019 07 03T10:01:14Z|INSDC status:public|Submitter Id:8|common name:zebrafish|dev stage:Shield|sample name:8|scientific name:Danio rerio                 NextSeq 500 sequencing ena EXPERIMENT Computational Biology Unit 22 08 2019 11:57:43:424 32 Shield 1 F19 None RCP seq   OTHER TRANSCRIPTOMIC other SINGLE ILLUMINA NextSeq 500   ERP116106 NextSeq 500 sequencing ENA FIRST PUBLIC:2020 03 27|ENA LAST UPDATE:2019 08 22     931166668.0 12252193.0 ena RUN Computational Biology Unit 22 08 2019 11:57:43:424 32 0:76 A:271081669;C:253947035;G:272354427;T:133774815;N:8722 76       271081669 253947035 272354427 133774815 8722 ERX3511295 ERS3556004 ERA2100634 Computational Biology Unit|European Nucleotide Archive Computational Biology Unit 1 0.15598   0.10707   0.99902   0.47314   76   B   usable mapping rate illumina nextseq unknown other unknown bulk unknown unknown   Unknown 2019-07-03 Gastrula Embryo Undetermined Embryo Imprecise
9731 9731 ERR3489879 ERX3511294 ERS3556004 ERP116106 PRJEB33323 Deconstructing the individual steps of vertebrate translation initiation ena-STUDY-Computational Biology Unit-03-07-2019-10:11:34:314-422 Other In eukaryotes the number of ribosomes synthesizing a given protein depends on how many are recruited to its mRNA their success in navigating its five prime untranslated region UTR and whether they recognize its start codon. Initiation of translation is a rate limiting step in protein synthesis and key to gene expression control 1 but despite this centrality it remains poorly understood 2 3. Here we introduce ribosome complex profiling RCP seq to capture the transcriptome wide occupancy of scanning initiating elongating and terminating ribosome complexes in a higher eukaryote. We track scanning and elongating ribosomes across all five prime UTRs in zebrafish which enable us to assess the individual regulatory contributions from the three stages of initiation: ribosome recruitment scanning of the five prime UTR and recognition of the start codon. Our data sheds light on small subunit recruitment to mRNAs presenting evidence for the threading model and demonstrates that sequence features regulate this recruitment. We estimate the processivity of scanning ribosomes as they traverse the five prime UTR and show that the repressive effects of upstream open reading frames depend on the efficiency of both translation initiation and termination. Finally we determine the optimal initiation contexts by directly estimating the conversion of scanning to elongating ribosomes and demonstrate specific regulation of translation initiation at the endoplasmic reticulum. Our results open for the possibility of deconvoluting translation initiation into separate stages and provides the first view of global occupancy of ribosomal small subunits in a vertebrate. ENA FIRST PUBLIC:2020 03 27|ENA LAST UPDATE:2019 07 03     Shield 1 SAMEA5752545 Computational Biology Unit ENA FIRST PUBLIC:2020 03 27T17:04:59Z|ENA LAST UPDATE:2019 07 03T10:01:14Z|External Id:SAMEA5752545|INSDC center name:Computational Biology Unit|INSDC first public:2020 03 27T17:04:59Z|INSDC last update:2019 07 03T10:01:14Z|INSDC status:public|Submitter Id:8|common name:zebrafish|dev stage:Shield|sample name:8|scientific name:Danio rerio                 NextSeq 500 sequencing ena EXPERIMENT Computational Biology Unit 22 08 2019 11:57:43:424 31 Shield 1 F18 None RCP seq   OTHER TRANSCRIPTOMIC other SINGLE ILLUMINA NextSeq 500   ERP116106 NextSeq 500 sequencing ENA FIRST PUBLIC:2020 03 27|ENA LAST UPDATE:2019 08 22     1506513268.0 19822543.0 ena RUN Computational Biology Unit 22 08 2019 11:57:43:424 31 0:76 A:493273515;C:456544968;G:391677033;T:165002559;N:15193 76       493273515 456544968 391677033 165002559 15193 ERX3511294 ERS3556004 ERA2100634 Computational Biology Unit|European Nucleotide Archive Computational Biology Unit 1 0.01562   0.0053   0.99908   0.8127   76   B   usable mapping rate illumina nextseq unknown other unknown bulk unknown unknown   Unknown 2019-07-03 Gastrula Embryo Undetermined Embryo Imprecise
9732 9732 ERR3489878 ERX3511293 ERS3556004 ERP116106 PRJEB33323 Deconstructing the individual steps of vertebrate translation initiation ena-STUDY-Computational Biology Unit-03-07-2019-10:11:34:314-422 Other In eukaryotes the number of ribosomes synthesizing a given protein depends on how many are recruited to its mRNA their success in navigating its five prime untranslated region UTR and whether they recognize its start codon. Initiation of translation is a rate limiting step in protein synthesis and key to gene expression control 1 but despite this centrality it remains poorly understood 2 3. Here we introduce ribosome complex profiling RCP seq to capture the transcriptome wide occupancy of scanning initiating elongating and terminating ribosome complexes in a higher eukaryote. We track scanning and elongating ribosomes across all five prime UTRs in zebrafish which enable us to assess the individual regulatory contributions from the three stages of initiation: ribosome recruitment scanning of the five prime UTR and recognition of the start codon. Our data sheds light on small subunit recruitment to mRNAs presenting evidence for the threading model and demonstrates that sequence features regulate this recruitment. We estimate the processivity of scanning ribosomes as they traverse the five prime UTR and show that the repressive effects of upstream open reading frames depend on the efficiency of both translation initiation and termination. Finally we determine the optimal initiation contexts by directly estimating the conversion of scanning to elongating ribosomes and demonstrate specific regulation of translation initiation at the endoplasmic reticulum. Our results open for the possibility of deconvoluting translation initiation into separate stages and provides the first view of global occupancy of ribosomal small subunits in a vertebrate. ENA FIRST PUBLIC:2020 03 27|ENA LAST UPDATE:2019 07 03     Shield 1 SAMEA5752545 Computational Biology Unit ENA FIRST PUBLIC:2020 03 27T17:04:59Z|ENA LAST UPDATE:2019 07 03T10:01:14Z|External Id:SAMEA5752545|INSDC center name:Computational Biology Unit|INSDC first public:2020 03 27T17:04:59Z|INSDC last update:2019 07 03T10:01:14Z|INSDC status:public|Submitter Id:8|common name:zebrafish|dev stage:Shield|sample name:8|scientific name:Danio rerio                 NextSeq 500 sequencing ena EXPERIMENT Computational Biology Unit 22 08 2019 11:57:43:424 30 Shield 1 F17 None RCP seq   OTHER TRANSCRIPTOMIC other SINGLE ILLUMINA NextSeq 500   ERP116106 NextSeq 500 sequencing ENA FIRST PUBLIC:2020 03 27|ENA LAST UPDATE:2019 08 22     1259456496.0 16571796.0 ena RUN Computational Biology Unit 22 08 2019 11:57:43:424 30 0:76 A:471473123;C:333726472;G:302016190;T:152228002;N:12709 76       471473123 333726472 302016190 152228002 12709 ERX3511293 ERS3556004 ERA2100634 Computational Biology Unit|European Nucleotide Archive Computational Biology Unit 1 0.18339   0.12544   0.99928   0.22368   76   B   usable mapping rate illumina nextseq unknown other unknown bulk unknown unknown   Unknown 2019-07-03 Gastrula Embryo Undetermined Embryo Imprecise
9733 9733 ERR3489877 ERX3511292 ERS3556004 ERP116106 PRJEB33323 Deconstructing the individual steps of vertebrate translation initiation ena-STUDY-Computational Biology Unit-03-07-2019-10:11:34:314-422 Other In eukaryotes the number of ribosomes synthesizing a given protein depends on how many are recruited to its mRNA their success in navigating its five prime untranslated region UTR and whether they recognize its start codon. Initiation of translation is a rate limiting step in protein synthesis and key to gene expression control 1 but despite this centrality it remains poorly understood 2 3. Here we introduce ribosome complex profiling RCP seq to capture the transcriptome wide occupancy of scanning initiating elongating and terminating ribosome complexes in a higher eukaryote. We track scanning and elongating ribosomes across all five prime UTRs in zebrafish which enable us to assess the individual regulatory contributions from the three stages of initiation: ribosome recruitment scanning of the five prime UTR and recognition of the start codon. Our data sheds light on small subunit recruitment to mRNAs presenting evidence for the threading model and demonstrates that sequence features regulate this recruitment. We estimate the processivity of scanning ribosomes as they traverse the five prime UTR and show that the repressive effects of upstream open reading frames depend on the efficiency of both translation initiation and termination. Finally we determine the optimal initiation contexts by directly estimating the conversion of scanning to elongating ribosomes and demonstrate specific regulation of translation initiation at the endoplasmic reticulum. Our results open for the possibility of deconvoluting translation initiation into separate stages and provides the first view of global occupancy of ribosomal small subunits in a vertebrate. ENA FIRST PUBLIC:2020 03 27|ENA LAST UPDATE:2019 07 03     Shield 1 SAMEA5752545 Computational Biology Unit ENA FIRST PUBLIC:2020 03 27T17:04:59Z|ENA LAST UPDATE:2019 07 03T10:01:14Z|External Id:SAMEA5752545|INSDC center name:Computational Biology Unit|INSDC first public:2020 03 27T17:04:59Z|INSDC last update:2019 07 03T10:01:14Z|INSDC status:public|Submitter Id:8|common name:zebrafish|dev stage:Shield|sample name:8|scientific name:Danio rerio                 NextSeq 500 sequencing ena EXPERIMENT Computational Biology Unit 22 08 2019 11:57:43:424 29 Shield 1 F16 None RCP seq   OTHER TRANSCRIPTOMIC other SINGLE ILLUMINA NextSeq 500   ERP116106 NextSeq 500 sequencing ENA FIRST PUBLIC:2020 03 27|ENA LAST UPDATE:2019 08 22     1364615872.0 17955472.0 ena RUN Computational Biology Unit 22 08 2019 11:57:43:424 29 0:76 A:539462776;C:341141880;G:314571683;T:169426048;N:13485 76       539462776 341141880 314571683 169426048 13485 ERX3511292 ERS3556004 ERA2100634 Computational Biology Unit|European Nucleotide Archive Computational Biology Unit 1 0.11663   0.07319   0.99939   0.25377   76   B   usable mapping rate illumina nextseq unknown other unknown bulk unknown unknown   Unknown 2019-07-03 Gastrula Embryo Undetermined Embryo Imprecise
9734 9734 ERR3489876 ERX3511291 ERS3556004 ERP116106 PRJEB33323 Deconstructing the individual steps of vertebrate translation initiation ena-STUDY-Computational Biology Unit-03-07-2019-10:11:34:314-422 Other In eukaryotes the number of ribosomes synthesizing a given protein depends on how many are recruited to its mRNA their success in navigating its five prime untranslated region UTR and whether they recognize its start codon. Initiation of translation is a rate limiting step in protein synthesis and key to gene expression control 1 but despite this centrality it remains poorly understood 2 3. Here we introduce ribosome complex profiling RCP seq to capture the transcriptome wide occupancy of scanning initiating elongating and terminating ribosome complexes in a higher eukaryote. We track scanning and elongating ribosomes across all five prime UTRs in zebrafish which enable us to assess the individual regulatory contributions from the three stages of initiation: ribosome recruitment scanning of the five prime UTR and recognition of the start codon. Our data sheds light on small subunit recruitment to mRNAs presenting evidence for the threading model and demonstrates that sequence features regulate this recruitment. We estimate the processivity of scanning ribosomes as they traverse the five prime UTR and show that the repressive effects of upstream open reading frames depend on the efficiency of both translation initiation and termination. Finally we determine the optimal initiation contexts by directly estimating the conversion of scanning to elongating ribosomes and demonstrate specific regulation of translation initiation at the endoplasmic reticulum. Our results open for the possibility of deconvoluting translation initiation into separate stages and provides the first view of global occupancy of ribosomal small subunits in a vertebrate. ENA FIRST PUBLIC:2020 03 27|ENA LAST UPDATE:2019 07 03     Shield 1 SAMEA5752545 Computational Biology Unit ENA FIRST PUBLIC:2020 03 27T17:04:59Z|ENA LAST UPDATE:2019 07 03T10:01:14Z|External Id:SAMEA5752545|INSDC center name:Computational Biology Unit|INSDC first public:2020 03 27T17:04:59Z|INSDC last update:2019 07 03T10:01:14Z|INSDC status:public|Submitter Id:8|common name:zebrafish|dev stage:Shield|sample name:8|scientific name:Danio rerio                 NextSeq 500 sequencing ena EXPERIMENT Computational Biology Unit 22 08 2019 11:57:43:424 28 Shield 1 F15 None RCP seq   OTHER TRANSCRIPTOMIC other SINGLE ILLUMINA NextSeq 500   ERP116106 NextSeq 500 sequencing ENA FIRST PUBLIC:2020 03 27|ENA LAST UPDATE:2019 08 22     952605888.0 12534288.0 ena RUN Computational Biology Unit 22 08 2019 11:57:43:424 28 0:76 A:414133320;C:219437277;G:207418049;T:111607418;N:9824 76       414133320 219437277 207418049 111607418 9824 ERX3511291 ERS3556004 ERA2100634 Computational Biology Unit|European Nucleotide Archive Computational Biology Unit 1 0.17603   0.10972   0.99935   0.13311   76   B   usable mapping rate illumina nextseq unknown other unknown bulk unknown unknown   Unknown 2019-07-03 Gastrula Embryo Undetermined Embryo Imprecise
9735 9735 ERR3489875 ERX3511290 ERS3556004 ERP116106 PRJEB33323 Deconstructing the individual steps of vertebrate translation initiation ena-STUDY-Computational Biology Unit-03-07-2019-10:11:34:314-422 Other In eukaryotes the number of ribosomes synthesizing a given protein depends on how many are recruited to its mRNA their success in navigating its five prime untranslated region UTR and whether they recognize its start codon. Initiation of translation is a rate limiting step in protein synthesis and key to gene expression control 1 but despite this centrality it remains poorly understood 2 3. Here we introduce ribosome complex profiling RCP seq to capture the transcriptome wide occupancy of scanning initiating elongating and terminating ribosome complexes in a higher eukaryote. We track scanning and elongating ribosomes across all five prime UTRs in zebrafish which enable us to assess the individual regulatory contributions from the three stages of initiation: ribosome recruitment scanning of the five prime UTR and recognition of the start codon. Our data sheds light on small subunit recruitment to mRNAs presenting evidence for the threading model and demonstrates that sequence features regulate this recruitment. We estimate the processivity of scanning ribosomes as they traverse the five prime UTR and show that the repressive effects of upstream open reading frames depend on the efficiency of both translation initiation and termination. Finally we determine the optimal initiation contexts by directly estimating the conversion of scanning to elongating ribosomes and demonstrate specific regulation of translation initiation at the endoplasmic reticulum. Our results open for the possibility of deconvoluting translation initiation into separate stages and provides the first view of global occupancy of ribosomal small subunits in a vertebrate. ENA FIRST PUBLIC:2020 03 27|ENA LAST UPDATE:2019 07 03     Shield 1 SAMEA5752545 Computational Biology Unit ENA FIRST PUBLIC:2020 03 27T17:04:59Z|ENA LAST UPDATE:2019 07 03T10:01:14Z|External Id:SAMEA5752545|INSDC center name:Computational Biology Unit|INSDC first public:2020 03 27T17:04:59Z|INSDC last update:2019 07 03T10:01:14Z|INSDC status:public|Submitter Id:8|common name:zebrafish|dev stage:Shield|sample name:8|scientific name:Danio rerio                 NextSeq 500 sequencing ena EXPERIMENT Computational Biology Unit 22 08 2019 11:57:43:424 27 Shield 1 F14 None RCP seq   OTHER TRANSCRIPTOMIC other SINGLE ILLUMINA NextSeq 500   ERP116106 NextSeq 500 sequencing ENA FIRST PUBLIC:2020 03 27|ENA LAST UPDATE:2019 08 22     870952628.0 11459903.0 ena RUN Computational Biology Unit 22 08 2019 11:57:43:424 27 0:76 A:357710338;C:221475453;G:191231014;T:100526675;N:9148 76       357710338 221475453 191231014 100526675 9148 ERX3511290 ERS3556004 ERA2100634 Computational Biology Unit|European Nucleotide Archive Computational Biology Unit 1 0.1248   0.06422   0.99896   0.34819   76   B   usable mapping rate illumina nextseq unknown other unknown bulk unknown unknown   Unknown 2019-07-03 Gastrula Embryo Undetermined Embryo Imprecise
9736 9736 ERR3489874 ERX3511289 ERS3556004 ERP116106 PRJEB33323 Deconstructing the individual steps of vertebrate translation initiation ena-STUDY-Computational Biology Unit-03-07-2019-10:11:34:314-422 Other In eukaryotes the number of ribosomes synthesizing a given protein depends on how many are recruited to its mRNA their success in navigating its five prime untranslated region UTR and whether they recognize its start codon. Initiation of translation is a rate limiting step in protein synthesis and key to gene expression control 1 but despite this centrality it remains poorly understood 2 3. Here we introduce ribosome complex profiling RCP seq to capture the transcriptome wide occupancy of scanning initiating elongating and terminating ribosome complexes in a higher eukaryote. We track scanning and elongating ribosomes across all five prime UTRs in zebrafish which enable us to assess the individual regulatory contributions from the three stages of initiation: ribosome recruitment scanning of the five prime UTR and recognition of the start codon. Our data sheds light on small subunit recruitment to mRNAs presenting evidence for the threading model and demonstrates that sequence features regulate this recruitment. We estimate the processivity of scanning ribosomes as they traverse the five prime UTR and show that the repressive effects of upstream open reading frames depend on the efficiency of both translation initiation and termination. Finally we determine the optimal initiation contexts by directly estimating the conversion of scanning to elongating ribosomes and demonstrate specific regulation of translation initiation at the endoplasmic reticulum. Our results open for the possibility of deconvoluting translation initiation into separate stages and provides the first view of global occupancy of ribosomal small subunits in a vertebrate. ENA FIRST PUBLIC:2020 03 27|ENA LAST UPDATE:2019 07 03     Shield 1 SAMEA5752545 Computational Biology Unit ENA FIRST PUBLIC:2020 03 27T17:04:59Z|ENA LAST UPDATE:2019 07 03T10:01:14Z|External Id:SAMEA5752545|INSDC center name:Computational Biology Unit|INSDC first public:2020 03 27T17:04:59Z|INSDC last update:2019 07 03T10:01:14Z|INSDC status:public|Submitter Id:8|common name:zebrafish|dev stage:Shield|sample name:8|scientific name:Danio rerio                 NextSeq 500 sequencing ena EXPERIMENT Computational Biology Unit 22 08 2019 11:57:43:424 26 Shield 1 F13 None RCP seq   OTHER TRANSCRIPTOMIC other SINGLE ILLUMINA NextSeq 500   ERP116106 NextSeq 500 sequencing ENA FIRST PUBLIC:2020 03 27|ENA LAST UPDATE:2019 08 22     981672620.0 12916745.0 ena RUN Computational Biology Unit 22 08 2019 11:57:43:424 26 0:76 A:434153198;C:223317075;G:198260771;T:125932145;N:9431 76       434153198 223317075 198260771 125932145 9431 ERX3511289 ERS3556004 ERA2100634 Computational Biology Unit|European Nucleotide Archive Computational Biology Unit 1 0.44603   0.25602   0.99874   0.18074   76   B   usable mapping rate illumina nextseq unknown other unknown bulk unknown unknown   Unknown 2019-07-03 Gastrula Embryo Undetermined Embryo Imprecise
9737 9737 ERR3489873 ERX3511288 ERS3556004 ERP116106 PRJEB33323 Deconstructing the individual steps of vertebrate translation initiation ena-STUDY-Computational Biology Unit-03-07-2019-10:11:34:314-422 Other In eukaryotes the number of ribosomes synthesizing a given protein depends on how many are recruited to its mRNA their success in navigating its five prime untranslated region UTR and whether they recognize its start codon. Initiation of translation is a rate limiting step in protein synthesis and key to gene expression control 1 but despite this centrality it remains poorly understood 2 3. Here we introduce ribosome complex profiling RCP seq to capture the transcriptome wide occupancy of scanning initiating elongating and terminating ribosome complexes in a higher eukaryote. We track scanning and elongating ribosomes across all five prime UTRs in zebrafish which enable us to assess the individual regulatory contributions from the three stages of initiation: ribosome recruitment scanning of the five prime UTR and recognition of the start codon. Our data sheds light on small subunit recruitment to mRNAs presenting evidence for the threading model and demonstrates that sequence features regulate this recruitment. We estimate the processivity of scanning ribosomes as they traverse the five prime UTR and show that the repressive effects of upstream open reading frames depend on the efficiency of both translation initiation and termination. Finally we determine the optimal initiation contexts by directly estimating the conversion of scanning to elongating ribosomes and demonstrate specific regulation of translation initiation at the endoplasmic reticulum. Our results open for the possibility of deconvoluting translation initiation into separate stages and provides the first view of global occupancy of ribosomal small subunits in a vertebrate. ENA FIRST PUBLIC:2020 03 27|ENA LAST UPDATE:2019 07 03     Shield 1 SAMEA5752545 Computational Biology Unit ENA FIRST PUBLIC:2020 03 27T17:04:59Z|ENA LAST UPDATE:2019 07 03T10:01:14Z|External Id:SAMEA5752545|INSDC center name:Computational Biology Unit|INSDC first public:2020 03 27T17:04:59Z|INSDC last update:2019 07 03T10:01:14Z|INSDC status:public|Submitter Id:8|common name:zebrafish|dev stage:Shield|sample name:8|scientific name:Danio rerio                 NextSeq 500 sequencing ena EXPERIMENT Computational Biology Unit 22 08 2019 11:57:43:424 25 Shield 1 F12 None RCP seq   OTHER TRANSCRIPTOMIC other SINGLE ILLUMINA NextSeq 500   ERP116106 NextSeq 500 sequencing ENA FIRST PUBLIC:2020 03 27|ENA LAST UPDATE:2019 08 22     1304618128.0 17166028.0 ena RUN Computational Biology Unit 22 08 2019 11:57:43:424 25 0:76 A:651341516;C:270458496;G:243929520;T:138874830;N:13766 76       651341516 270458496 243929520 138874830 13766 ERX3511288 ERS3556004 ERA2100634 Computational Biology Unit|European Nucleotide Archive Computational Biology Unit 1 0.64293   0.38159   0.99886   0.07313   76   B   usable mapping rate illumina nextseq unknown other unknown bulk unknown unknown   Unknown 2019-07-03 Gastrula Embryo Undetermined Embryo Imprecise
9738 9738 ERR3489872 ERX3511287 ERS3556004 ERP116106 PRJEB33323 Deconstructing the individual steps of vertebrate translation initiation ena-STUDY-Computational Biology Unit-03-07-2019-10:11:34:314-422 Other In eukaryotes the number of ribosomes synthesizing a given protein depends on how many are recruited to its mRNA their success in navigating its five prime untranslated region UTR and whether they recognize its start codon. Initiation of translation is a rate limiting step in protein synthesis and key to gene expression control 1 but despite this centrality it remains poorly understood 2 3. Here we introduce ribosome complex profiling RCP seq to capture the transcriptome wide occupancy of scanning initiating elongating and terminating ribosome complexes in a higher eukaryote. We track scanning and elongating ribosomes across all five prime UTRs in zebrafish which enable us to assess the individual regulatory contributions from the three stages of initiation: ribosome recruitment scanning of the five prime UTR and recognition of the start codon. Our data sheds light on small subunit recruitment to mRNAs presenting evidence for the threading model and demonstrates that sequence features regulate this recruitment. We estimate the processivity of scanning ribosomes as they traverse the five prime UTR and show that the repressive effects of upstream open reading frames depend on the efficiency of both translation initiation and termination. Finally we determine the optimal initiation contexts by directly estimating the conversion of scanning to elongating ribosomes and demonstrate specific regulation of translation initiation at the endoplasmic reticulum. Our results open for the possibility of deconvoluting translation initiation into separate stages and provides the first view of global occupancy of ribosomal small subunits in a vertebrate. ENA FIRST PUBLIC:2020 03 27|ENA LAST UPDATE:2019 07 03     Shield 1 SAMEA5752545 Computational Biology Unit ENA FIRST PUBLIC:2020 03 27T17:04:59Z|ENA LAST UPDATE:2019 07 03T10:01:14Z|External Id:SAMEA5752545|INSDC center name:Computational Biology Unit|INSDC first public:2020 03 27T17:04:59Z|INSDC last update:2019 07 03T10:01:14Z|INSDC status:public|Submitter Id:8|common name:zebrafish|dev stage:Shield|sample name:8|scientific name:Danio rerio                 NextSeq 500 sequencing ena EXPERIMENT Computational Biology Unit 22 08 2019 11:57:43:424 24 Shield 1 F10 None RCP seq   OTHER TRANSCRIPTOMIC other SINGLE ILLUMINA NextSeq 500   ERP116106 NextSeq 500 sequencing ENA FIRST PUBLIC:2020 03 27|ENA LAST UPDATE:2019 08 22     1336115948.0 17580473.0 ena RUN Computational Biology Unit 22 08 2019 11:57:43:424 24 0:76 A:608634206;C:295943144;G:286494431;T:145029697;N:14470 76       608634206 295943144 286494431 145029697 14470 ERX3511287 ERS3556004 ERA2100634 Computational Biology Unit|European Nucleotide Archive Computational Biology Unit 1 0.68758   0.47517   0.99898   0.02301   76   B   usable mapping rate illumina nextseq unknown other unknown bulk unknown unknown   Unknown 2019-07-03 Gastrula Embryo Undetermined Embryo Imprecise
9739 9739 ERR3489871 ERX3511286 ERS3556004 ERP116106 PRJEB33323 Deconstructing the individual steps of vertebrate translation initiation ena-STUDY-Computational Biology Unit-03-07-2019-10:11:34:314-422 Other In eukaryotes the number of ribosomes synthesizing a given protein depends on how many are recruited to its mRNA their success in navigating its five prime untranslated region UTR and whether they recognize its start codon. Initiation of translation is a rate limiting step in protein synthesis and key to gene expression control 1 but despite this centrality it remains poorly understood 2 3. Here we introduce ribosome complex profiling RCP seq to capture the transcriptome wide occupancy of scanning initiating elongating and terminating ribosome complexes in a higher eukaryote. We track scanning and elongating ribosomes across all five prime UTRs in zebrafish which enable us to assess the individual regulatory contributions from the three stages of initiation: ribosome recruitment scanning of the five prime UTR and recognition of the start codon. Our data sheds light on small subunit recruitment to mRNAs presenting evidence for the threading model and demonstrates that sequence features regulate this recruitment. We estimate the processivity of scanning ribosomes as they traverse the five prime UTR and show that the repressive effects of upstream open reading frames depend on the efficiency of both translation initiation and termination. Finally we determine the optimal initiation contexts by directly estimating the conversion of scanning to elongating ribosomes and demonstrate specific regulation of translation initiation at the endoplasmic reticulum. Our results open for the possibility of deconvoluting translation initiation into separate stages and provides the first view of global occupancy of ribosomal small subunits in a vertebrate. ENA FIRST PUBLIC:2020 03 27|ENA LAST UPDATE:2019 07 03     Shield 1 SAMEA5752545 Computational Biology Unit ENA FIRST PUBLIC:2020 03 27T17:04:59Z|ENA LAST UPDATE:2019 07 03T10:01:14Z|External Id:SAMEA5752545|INSDC center name:Computational Biology Unit|INSDC first public:2020 03 27T17:04:59Z|INSDC last update:2019 07 03T10:01:14Z|INSDC status:public|Submitter Id:8|common name:zebrafish|dev stage:Shield|sample name:8|scientific name:Danio rerio                 NextSeq 500 sequencing ena EXPERIMENT Computational Biology Unit 22 08 2019 11:57:43:424 23 Shield 1 F9 None RCP seq   OTHER TRANSCRIPTOMIC other SINGLE ILLUMINA NextSeq 500   ERP116106 NextSeq 500 sequencing ENA FIRST PUBLIC:2020 03 27|ENA LAST UPDATE:2019 08 22     1434402492.0 18873717.0 ena RUN Computational Biology Unit 22 08 2019 11:57:43:424 23 0:76 A:658705664;C:297967081;G:295595736;T:182118632;N:15379 76       658705664 297967081 295595736 182118632 15379 ERX3511286 ERS3556004 ERA2100634 Computational Biology Unit|European Nucleotide Archive Computational Biology Unit 1 0.74246   0.44235   0.99701   0.03112   76   B   usable mapping rate illumina nextseq unknown other unknown bulk unknown unknown   Unknown 2019-07-03 Gastrula Embryo Undetermined Embryo Imprecise
9740 9740 ERR3489870 ERX3511285 ERS3556007 ERP116106 PRJEB33323 Deconstructing the individual steps of vertebrate translation initiation ena-STUDY-Computational Biology Unit-03-07-2019-10:11:34:314-422 Other In eukaryotes the number of ribosomes synthesizing a given protein depends on how many are recruited to its mRNA their success in navigating its five prime untranslated region UTR and whether they recognize its start codon. Initiation of translation is a rate limiting step in protein synthesis and key to gene expression control 1 but despite this centrality it remains poorly understood 2 3. Here we introduce ribosome complex profiling RCP seq to capture the transcriptome wide occupancy of scanning initiating elongating and terminating ribosome complexes in a higher eukaryote. We track scanning and elongating ribosomes across all five prime UTRs in zebrafish which enable us to assess the individual regulatory contributions from the three stages of initiation: ribosome recruitment scanning of the five prime UTR and recognition of the start codon. Our data sheds light on small subunit recruitment to mRNAs presenting evidence for the threading model and demonstrates that sequence features regulate this recruitment. We estimate the processivity of scanning ribosomes as they traverse the five prime UTR and show that the repressive effects of upstream open reading frames depend on the efficiency of both translation initiation and termination. Finally we determine the optimal initiation contexts by directly estimating the conversion of scanning to elongating ribosomes and demonstrate specific regulation of translation initiation at the endoplasmic reticulum. Our results open for the possibility of deconvoluting translation initiation into separate stages and provides the first view of global occupancy of ribosomal small subunits in a vertebrate. ENA FIRST PUBLIC:2020 03 27|ENA LAST UPDATE:2019 07 03     Shield 4150NT SAMEA5752548 Computational Biology Unit ENA FIRST PUBLIC:2020 03 27T17:04:59Z|ENA LAST UPDATE:2019 07 03T10:01:14Z|External Id:SAMEA5752548|INSDC center name:Computational Biology Unit|INSDC first public:2020 03 27T17:04:59Z|INSDC last update:2019 07 03T10:01:14Z|INSDC status:public|Submitter Id:11|common name:zebrafish|dev stage:Shield|sample name:11|scientific name:Danio rerio                 NextSeq 500 sequencing ena EXPERIMENT Computational Biology Unit 22 08 2019 11:57:43:424 22 Shield 4150NT LSU None RCP seq   OTHER TRANSCRIPTOMIC other SINGLE ILLUMINA NextSeq 500   ERP116106 NextSeq 500 sequencing ENA FIRST PUBLIC:2020 03 27|ENA LAST UPDATE:2019 08 24     24063208094.0 159358994.0 ena RUN Computational Biology Unit 22 08 2019 11:57:43:424 22 0:151 A:7153064588;C:5242791119;G:8513736630;T:3152547276;N:1068481 151       7153064588 5242791119 8513736630 3152547276 1068481 ERX3511285 ERS3556007 ERA2100634 Computational Biology Unit|European Nucleotide Archive Computational Biology Unit 1 0.81267   0.27138   0.99868   0.91938   151   B   usable mapping rate illumina nextseq unknown other unknown bulk unknown unknown   Unknown 2019-07-03 Gastrula Embryo Undetermined Embryo Imprecise
9741 9741 ERR3489869 ERX3511284 ERS3556007 ERP116106 PRJEB33323 Deconstructing the individual steps of vertebrate translation initiation ena-STUDY-Computational Biology Unit-03-07-2019-10:11:34:314-422 Other In eukaryotes the number of ribosomes synthesizing a given protein depends on how many are recruited to its mRNA their success in navigating its five prime untranslated region UTR and whether they recognize its start codon. Initiation of translation is a rate limiting step in protein synthesis and key to gene expression control 1 but despite this centrality it remains poorly understood 2 3. Here we introduce ribosome complex profiling RCP seq to capture the transcriptome wide occupancy of scanning initiating elongating and terminating ribosome complexes in a higher eukaryote. We track scanning and elongating ribosomes across all five prime UTRs in zebrafish which enable us to assess the individual regulatory contributions from the three stages of initiation: ribosome recruitment scanning of the five prime UTR and recognition of the start codon. Our data sheds light on small subunit recruitment to mRNAs presenting evidence for the threading model and demonstrates that sequence features regulate this recruitment. We estimate the processivity of scanning ribosomes as they traverse the five prime UTR and show that the repressive effects of upstream open reading frames depend on the efficiency of both translation initiation and termination. Finally we determine the optimal initiation contexts by directly estimating the conversion of scanning to elongating ribosomes and demonstrate specific regulation of translation initiation at the endoplasmic reticulum. Our results open for the possibility of deconvoluting translation initiation into separate stages and provides the first view of global occupancy of ribosomal small subunits in a vertebrate. ENA FIRST PUBLIC:2020 03 27|ENA LAST UPDATE:2019 07 03     Shield 4150NT SAMEA5752548 Computational Biology Unit ENA FIRST PUBLIC:2020 03 27T17:04:59Z|ENA LAST UPDATE:2019 07 03T10:01:14Z|External Id:SAMEA5752548|INSDC center name:Computational Biology Unit|INSDC first public:2020 03 27T17:04:59Z|INSDC last update:2019 07 03T10:01:14Z|INSDC status:public|Submitter Id:11|common name:zebrafish|dev stage:Shield|sample name:11|scientific name:Danio rerio                 NextSeq 500 sequencing ena EXPERIMENT Computational Biology Unit 22 08 2019 11:57:43:424 21 Shield 4150NT SSU None RCP seq   OTHER TRANSCRIPTOMIC other SINGLE ILLUMINA NextSeq 500   ERP116106 NextSeq 500 sequencing ENA FIRST PUBLIC:2020 03 27|ENA LAST UPDATE:2019 08 24     14987998619.0 99258269.0 ena RUN Computational Biology Unit 22 08 2019 11:57:43:424 21 0:151 A:4578051806;C:2616328922;G:5914185813;T:1878780090;N:651988 151       4578051806 2616328922 5914185813 1878780090 651988 ERX3511284 ERS3556007 ERA2100634 Computational Biology Unit|European Nucleotide Archive Computational Biology Unit 1 0.77096   0.5278   0.99833   0.42635   151   B   usable mapping rate illumina nextseq unknown other unknown bulk unknown unknown   Unknown 2019-07-03 Gastrula Embryo Undetermined Embryo Imprecise
9742 9742 ERR3489868 ERX3511283 ERS3556003 ERP116106 PRJEB33323 Deconstructing the individual steps of vertebrate translation initiation ena-STUDY-Computational Biology Unit-03-07-2019-10:11:34:314-422 Other In eukaryotes the number of ribosomes synthesizing a given protein depends on how many are recruited to its mRNA their success in navigating its five prime untranslated region UTR and whether they recognize its start codon. Initiation of translation is a rate limiting step in protein synthesis and key to gene expression control 1 but despite this centrality it remains poorly understood 2 3. Here we introduce ribosome complex profiling RCP seq to capture the transcriptome wide occupancy of scanning initiating elongating and terminating ribosome complexes in a higher eukaryote. We track scanning and elongating ribosomes across all five prime UTRs in zebrafish which enable us to assess the individual regulatory contributions from the three stages of initiation: ribosome recruitment scanning of the five prime UTR and recognition of the start codon. Our data sheds light on small subunit recruitment to mRNAs presenting evidence for the threading model and demonstrates that sequence features regulate this recruitment. We estimate the processivity of scanning ribosomes as they traverse the five prime UTR and show that the repressive effects of upstream open reading frames depend on the efficiency of both translation initiation and termination. Finally we determine the optimal initiation contexts by directly estimating the conversion of scanning to elongating ribosomes and demonstrate specific regulation of translation initiation at the endoplasmic reticulum. Our results open for the possibility of deconvoluting translation initiation into separate stages and provides the first view of global occupancy of ribosomal small subunits in a vertebrate. ENA FIRST PUBLIC:2020 03 27|ENA LAST UPDATE:2019 07 03     64 cell 4Ei 10 SAMEA5752544 Computational Biology Unit ENA FIRST PUBLIC:2020 03 27T17:04:59Z|ENA LAST UPDATE:2019 07 03T10:01:14Z|External Id:SAMEA5752544|INSDC center name:Computational Biology Unit|INSDC first public:2020 03 27T17:04:59Z|INSDC last update:2019 07 03T10:01:14Z|INSDC status:public|Submitter Id:7|common name:zebrafish|dev stage:64 cell|sample name:7|scientific name:Danio rerio                 NextSeq 500 sequencing ena EXPERIMENT Computational Biology Unit 22 08 2019 11:57:43:424 20 64 cell 4Ei 10 SSU None RCP seq   OTHER TRANSCRIPTOMIC other SINGLE ILLUMINA NextSeq 500   ERP116106 NextSeq 500 sequencing ENA FIRST PUBLIC:2020 03 27|ENA LAST UPDATE:2019 08 22     5928054796.0 78000721.0 ena RUN Computational Biology Unit 22 08 2019 11:57:43:424 20 0:76 A:2109854905;C:1377083508;G:1616166285;T:824889630;N:60468 76       2109854905 1377083508 1616166285 824889630 60468 ERX3511283 ERS3556003 ERA2100634 Computational Biology Unit|European Nucleotide Archive Computational Biology Unit 1 0.61525   0.45233   0.99379   0.57373   76   B   usable mapping rate illumina nextseq unknown other unknown bulk unknown unknown   Unknown 2019-07-03 Cleavage Embryo Undetermined Embryo Imprecise
9743 9743 ERR3489867 ERX3511282 ERS3556003 ERP116106 PRJEB33323 Deconstructing the individual steps of vertebrate translation initiation ena-STUDY-Computational Biology Unit-03-07-2019-10:11:34:314-422 Other In eukaryotes the number of ribosomes synthesizing a given protein depends on how many are recruited to its mRNA their success in navigating its five prime untranslated region UTR and whether they recognize its start codon. Initiation of translation is a rate limiting step in protein synthesis and key to gene expression control 1 but despite this centrality it remains poorly understood 2 3. Here we introduce ribosome complex profiling RCP seq to capture the transcriptome wide occupancy of scanning initiating elongating and terminating ribosome complexes in a higher eukaryote. We track scanning and elongating ribosomes across all five prime UTRs in zebrafish which enable us to assess the individual regulatory contributions from the three stages of initiation: ribosome recruitment scanning of the five prime UTR and recognition of the start codon. Our data sheds light on small subunit recruitment to mRNAs presenting evidence for the threading model and demonstrates that sequence features regulate this recruitment. We estimate the processivity of scanning ribosomes as they traverse the five prime UTR and show that the repressive effects of upstream open reading frames depend on the efficiency of both translation initiation and termination. Finally we determine the optimal initiation contexts by directly estimating the conversion of scanning to elongating ribosomes and demonstrate specific regulation of translation initiation at the endoplasmic reticulum. Our results open for the possibility of deconvoluting translation initiation into separate stages and provides the first view of global occupancy of ribosomal small subunits in a vertebrate. ENA FIRST PUBLIC:2020 03 27|ENA LAST UPDATE:2019 07 03     64 cell 4Ei 10 SAMEA5752544 Computational Biology Unit ENA FIRST PUBLIC:2020 03 27T17:04:59Z|ENA LAST UPDATE:2019 07 03T10:01:14Z|External Id:SAMEA5752544|INSDC center name:Computational Biology Unit|INSDC first public:2020 03 27T17:04:59Z|INSDC last update:2019 07 03T10:01:14Z|INSDC status:public|Submitter Id:7|common name:zebrafish|dev stage:64 cell|sample name:7|scientific name:Danio rerio                 NextSeq 500 sequencing ena EXPERIMENT Computational Biology Unit 22 08 2019 11:57:43:424 19 64 cell 4Ei 10 SSU None RCP seq   OTHER TRANSCRIPTOMIC other SINGLE ILLUMINA NextSeq 500   ERP116106 NextSeq 500 sequencing ENA FIRST PUBLIC:2020 03 27|ENA LAST UPDATE:2019 08 22     9772604780.0 128586905.0 ena RUN Computational Biology Unit 22 08 2019 11:57:43:424 19 0:76 A:3901301079;C:2223067879;G:2553338016;T:1094797648;N:100158 76       3901301079 2223067879 2553338016 1094797648 100158 ERX3511282 ERS3556003 ERA2100634 Computational Biology Unit|European Nucleotide Archive Computational Biology Unit 1 0.52103   0.25046   0.9861   0.64575   76   B   usable mapping rate illumina nextseq unknown other unknown bulk unknown unknown   Unknown 2019-07-03 Cleavage Embryo Undetermined Embryo Imprecise
9744 9744 ERR3489866 ERX3511281 ERS3556002 ERP116106 PRJEB33323 Deconstructing the individual steps of vertebrate translation initiation ena-STUDY-Computational Biology Unit-03-07-2019-10:11:34:314-422 Other In eukaryotes the number of ribosomes synthesizing a given protein depends on how many are recruited to its mRNA their success in navigating its five prime untranslated region UTR and whether they recognize its start codon. Initiation of translation is a rate limiting step in protein synthesis and key to gene expression control 1 but despite this centrality it remains poorly understood 2 3. Here we introduce ribosome complex profiling RCP seq to capture the transcriptome wide occupancy of scanning initiating elongating and terminating ribosome complexes in a higher eukaryote. We track scanning and elongating ribosomes across all five prime UTRs in zebrafish which enable us to assess the individual regulatory contributions from the three stages of initiation: ribosome recruitment scanning of the five prime UTR and recognition of the start codon. Our data sheds light on small subunit recruitment to mRNAs presenting evidence for the threading model and demonstrates that sequence features regulate this recruitment. We estimate the processivity of scanning ribosomes as they traverse the five prime UTR and show that the repressive effects of upstream open reading frames depend on the efficiency of both translation initiation and termination. Finally we determine the optimal initiation contexts by directly estimating the conversion of scanning to elongating ribosomes and demonstrate specific regulation of translation initiation at the endoplasmic reticulum. Our results open for the possibility of deconvoluting translation initiation into separate stages and provides the first view of global occupancy of ribosomal small subunits in a vertebrate. ENA FIRST PUBLIC:2020 03 27|ENA LAST UPDATE:2019 07 03     64 cell 4Ei 0.1 SAMEA5752543 Computational Biology Unit ENA FIRST PUBLIC:2020 03 27T17:04:59Z|ENA LAST UPDATE:2019 07 03T10:01:14Z|External Id:SAMEA5752543|INSDC center name:Computational Biology Unit|INSDC first public:2020 03 27T17:04:59Z|INSDC last update:2019 07 03T10:01:14Z|INSDC status:public|Submitter Id:6|common name:zebrafish|dev stage:64 cell|sample name:6|scientific name:Danio rerio                 NextSeq 500 sequencing ena EXPERIMENT Computational Biology Unit 22 08 2019 11:57:43:424 18 64 cell 4Ei 0.1 LSU None RCP seq   OTHER TRANSCRIPTOMIC other SINGLE ILLUMINA NextSeq 500   ERP116106 NextSeq 500 sequencing ENA FIRST PUBLIC:2020 03 27|ENA LAST UPDATE:2019 09 26     6730725680.0 88562180.0 ena RUN Computational Biology Unit 22 08 2019 11:57:43:424 18 0:76 A:3805722011;C:1188715051;G:1382258076;T:353814168;N:216374 76       3805722011 1188715051 1382258076 353814168 216374 ERX3511281 ERS3556002 ERA2100634 Computational Biology Unit|European Nucleotide Archive Computational Biology Unit 1 0.61922   0.37217   0.99527   0.29148   76   B   usable mapping rate illumina nextseq unknown other unknown bulk unknown unknown   Unknown 2019-07-03 Cleavage Embryo Undetermined Embryo Imprecise
9745 9745 ERR3489865 ERX3511280 ERS3556002 ERP116106 PRJEB33323 Deconstructing the individual steps of vertebrate translation initiation ena-STUDY-Computational Biology Unit-03-07-2019-10:11:34:314-422 Other In eukaryotes the number of ribosomes synthesizing a given protein depends on how many are recruited to its mRNA their success in navigating its five prime untranslated region UTR and whether they recognize its start codon. Initiation of translation is a rate limiting step in protein synthesis and key to gene expression control 1 but despite this centrality it remains poorly understood 2 3. Here we introduce ribosome complex profiling RCP seq to capture the transcriptome wide occupancy of scanning initiating elongating and terminating ribosome complexes in a higher eukaryote. We track scanning and elongating ribosomes across all five prime UTRs in zebrafish which enable us to assess the individual regulatory contributions from the three stages of initiation: ribosome recruitment scanning of the five prime UTR and recognition of the start codon. Our data sheds light on small subunit recruitment to mRNAs presenting evidence for the threading model and demonstrates that sequence features regulate this recruitment. We estimate the processivity of scanning ribosomes as they traverse the five prime UTR and show that the repressive effects of upstream open reading frames depend on the efficiency of both translation initiation and termination. Finally we determine the optimal initiation contexts by directly estimating the conversion of scanning to elongating ribosomes and demonstrate specific regulation of translation initiation at the endoplasmic reticulum. Our results open for the possibility of deconvoluting translation initiation into separate stages and provides the first view of global occupancy of ribosomal small subunits in a vertebrate. ENA FIRST PUBLIC:2020 03 27|ENA LAST UPDATE:2019 07 03     64 cell 4Ei 0.1 SAMEA5752543 Computational Biology Unit ENA FIRST PUBLIC:2020 03 27T17:04:59Z|ENA LAST UPDATE:2019 07 03T10:01:14Z|External Id:SAMEA5752543|INSDC center name:Computational Biology Unit|INSDC first public:2020 03 27T17:04:59Z|INSDC last update:2019 07 03T10:01:14Z|INSDC status:public|Submitter Id:6|common name:zebrafish|dev stage:64 cell|sample name:6|scientific name:Danio rerio                 NextSeq 500 sequencing ena EXPERIMENT Computational Biology Unit 22 08 2019 11:57:43:424 17 64 cell 4Ei 0.1 SSU None RCP seq   OTHER TRANSCRIPTOMIC other SINGLE ILLUMINA NextSeq 500   ERP116106 NextSeq 500 sequencing ENA FIRST PUBLIC:2020 03 27|ENA LAST UPDATE:2019 09 26     10616304872.0 139688222.0 ena RUN Computational Biology Unit 22 08 2019 11:57:43:424 17 0:76 A:4717003484;C:2600725234;G:2586105174;T:712124440;N:346540 76       4717003484 2600725234 2586105174 712124440 346540 ERX3511280 ERS3556002 ERA2100634 Computational Biology Unit|European Nucleotide Archive Computational Biology Unit 1 0.30908   0.08252   0.94683   0.69548   76   B   usable mapping rate illumina nextseq unknown other unknown bulk unknown unknown   Unknown 2019-07-03 Cleavage Embryo Undetermined Embryo Imprecise
9746 9746 ERR3489864 ERX3511279 ERS3556006 ERP116106 PRJEB33323 Deconstructing the individual steps of vertebrate translation initiation ena-STUDY-Computational Biology Unit-03-07-2019-10:11:34:314-422 Other In eukaryotes the number of ribosomes synthesizing a given protein depends on how many are recruited to its mRNA their success in navigating its five prime untranslated region UTR and whether they recognize its start codon. Initiation of translation is a rate limiting step in protein synthesis and key to gene expression control 1 but despite this centrality it remains poorly understood 2 3. Here we introduce ribosome complex profiling RCP seq to capture the transcriptome wide occupancy of scanning initiating elongating and terminating ribosome complexes in a higher eukaryote. We track scanning and elongating ribosomes across all five prime UTRs in zebrafish which enable us to assess the individual regulatory contributions from the three stages of initiation: ribosome recruitment scanning of the five prime UTR and recognition of the start codon. Our data sheds light on small subunit recruitment to mRNAs presenting evidence for the threading model and demonstrates that sequence features regulate this recruitment. We estimate the processivity of scanning ribosomes as they traverse the five prime UTR and show that the repressive effects of upstream open reading frames depend on the efficiency of both translation initiation and termination. Finally we determine the optimal initiation contexts by directly estimating the conversion of scanning to elongating ribosomes and demonstrate specific regulation of translation initiation at the endoplasmic reticulum. Our results open for the possibility of deconvoluting translation initiation into separate stages and provides the first view of global occupancy of ribosomal small subunits in a vertebrate. ENA FIRST PUBLIC:2020 03 27|ENA LAST UPDATE:2019 07 03     Shield 3 SAMEA5752547 Computational Biology Unit ENA FIRST PUBLIC:2020 03 27T17:04:59Z|ENA LAST UPDATE:2019 07 03T10:01:14Z|External Id:SAMEA5752547|INSDC center name:Computational Biology Unit|INSDC first public:2020 03 27T17:04:59Z|INSDC last update:2019 07 03T10:01:14Z|INSDC status:public|Submitter Id:10|common name:zebrafish|dev stage:Shield|sample name:10|scientific name:Danio rerio                 NextSeq 500 sequencing ena EXPERIMENT Computational Biology Unit 22 08 2019 11:57:43:424 16 Shield 3 LSU None RCP seq   OTHER TRANSCRIPTOMIC other SINGLE ILLUMINA NextSeq 500   ERP116106 NextSeq 500 sequencing ENA FIRST PUBLIC:2020 03 27|ENA LAST UPDATE:2019 08 22     11777220072.0 154963422.0 ena RUN Computational Biology Unit 22 08 2019 11:57:43:424 16 0:76 A:3368038444;C:3190496935;G:3529701152;T:1688766119;N:217422 76       3368038444 3190496935 3529701152 1688766119 217422 ERX3511279 ERS3556006 ERA2100634 Computational Biology Unit|European Nucleotide Archive Computational Biology Unit 1 0.70681   0.18846   0.99332   0.71978   76   B   usable mapping rate illumina nextseq unknown other unknown bulk unknown unknown   Unknown 2019-07-03 Gastrula Embryo Undetermined Embryo Imprecise
9747 9747 ERR3489863 ERX3511278 ERS3556006 ERP116106 PRJEB33323 Deconstructing the individual steps of vertebrate translation initiation ena-STUDY-Computational Biology Unit-03-07-2019-10:11:34:314-422 Other In eukaryotes the number of ribosomes synthesizing a given protein depends on how many are recruited to its mRNA their success in navigating its five prime untranslated region UTR and whether they recognize its start codon. Initiation of translation is a rate limiting step in protein synthesis and key to gene expression control 1 but despite this centrality it remains poorly understood 2 3. Here we introduce ribosome complex profiling RCP seq to capture the transcriptome wide occupancy of scanning initiating elongating and terminating ribosome complexes in a higher eukaryote. We track scanning and elongating ribosomes across all five prime UTRs in zebrafish which enable us to assess the individual regulatory contributions from the three stages of initiation: ribosome recruitment scanning of the five prime UTR and recognition of the start codon. Our data sheds light on small subunit recruitment to mRNAs presenting evidence for the threading model and demonstrates that sequence features regulate this recruitment. We estimate the processivity of scanning ribosomes as they traverse the five prime UTR and show that the repressive effects of upstream open reading frames depend on the efficiency of both translation initiation and termination. Finally we determine the optimal initiation contexts by directly estimating the conversion of scanning to elongating ribosomes and demonstrate specific regulation of translation initiation at the endoplasmic reticulum. Our results open for the possibility of deconvoluting translation initiation into separate stages and provides the first view of global occupancy of ribosomal small subunits in a vertebrate. ENA FIRST PUBLIC:2020 03 27|ENA LAST UPDATE:2019 07 03     Shield 3 SAMEA5752547 Computational Biology Unit ENA FIRST PUBLIC:2020 03 27T17:04:59Z|ENA LAST UPDATE:2019 07 03T10:01:14Z|External Id:SAMEA5752547|INSDC center name:Computational Biology Unit|INSDC first public:2020 03 27T17:04:59Z|INSDC last update:2019 07 03T10:01:14Z|INSDC status:public|Submitter Id:10|common name:zebrafish|dev stage:Shield|sample name:10|scientific name:Danio rerio                 NextSeq 500 sequencing ena EXPERIMENT Computational Biology Unit 22 08 2019 11:57:43:424 15 Shield 3 SSU None RCP seq   OTHER TRANSCRIPTOMIC other SINGLE ILLUMINA NextSeq 500   ERP116106 NextSeq 500 sequencing ENA FIRST PUBLIC:2020 03 27|ENA LAST UPDATE:2019 08 22     7920419952.0 104216052.0 ena RUN Computational Biology Unit 22 08 2019 11:57:43:424 15 0:76 A:2990167185;C:1767708808;G:2104830363;T:1057568560;N:145036 76       2990167185 1767708808 2104830363 1057568560 145036 ERX3511278 ERS3556006 ERA2100634 Computational Biology Unit|European Nucleotide Archive Computational Biology Unit 1 0.5052   0.30841   0.99129   0.60948   76   B   usable mapping rate illumina nextseq unknown other unknown bulk unknown unknown   Unknown 2019-07-03 Gastrula Embryo Undetermined Embryo Imprecise
9748 9748 ERR3489862 ERX3511277 ERS3556005 ERP116106 PRJEB33323 Deconstructing the individual steps of vertebrate translation initiation ena-STUDY-Computational Biology Unit-03-07-2019-10:11:34:314-422 Other In eukaryotes the number of ribosomes synthesizing a given protein depends on how many are recruited to its mRNA their success in navigating its five prime untranslated region UTR and whether they recognize its start codon. Initiation of translation is a rate limiting step in protein synthesis and key to gene expression control 1 but despite this centrality it remains poorly understood 2 3. Here we introduce ribosome complex profiling RCP seq to capture the transcriptome wide occupancy of scanning initiating elongating and terminating ribosome complexes in a higher eukaryote. We track scanning and elongating ribosomes across all five prime UTRs in zebrafish which enable us to assess the individual regulatory contributions from the three stages of initiation: ribosome recruitment scanning of the five prime UTR and recognition of the start codon. Our data sheds light on small subunit recruitment to mRNAs presenting evidence for the threading model and demonstrates that sequence features regulate this recruitment. We estimate the processivity of scanning ribosomes as they traverse the five prime UTR and show that the repressive effects of upstream open reading frames depend on the efficiency of both translation initiation and termination. Finally we determine the optimal initiation contexts by directly estimating the conversion of scanning to elongating ribosomes and demonstrate specific regulation of translation initiation at the endoplasmic reticulum. Our results open for the possibility of deconvoluting translation initiation into separate stages and provides the first view of global occupancy of ribosomal small subunits in a vertebrate. ENA FIRST PUBLIC:2020 03 27|ENA LAST UPDATE:2019 07 03     Shield 2 SAMEA5752546 Computational Biology Unit ENA FIRST PUBLIC:2020 03 27T17:04:59Z|ENA LAST UPDATE:2019 07 03T10:01:14Z|External Id:SAMEA5752546|INSDC center name:Computational Biology Unit|INSDC first public:2020 03 27T17:04:59Z|INSDC last update:2019 07 03T10:01:14Z|INSDC status:public|Submitter Id:9|common name:zebrafish|dev stage:Shield|sample name:9|scientific name:Danio rerio                 NextSeq 500 sequencing ena EXPERIMENT Computational Biology Unit 22 08 2019 11:57:43:424 14 Shield 2 LSU None RCP seq   OTHER TRANSCRIPTOMIC other SINGLE ILLUMINA NextSeq 500   ERP116106 NextSeq 500 sequencing ENA FIRST PUBLIC:2020 03 27|ENA LAST UPDATE:2019 08 22     9775297004.0 128622329.0 ena RUN Computational Biology Unit 22 08 2019 11:57:43:424 14 0:76 A:4750565405;C:2540992255;G:1698817649;T:784832634;N:89061 76       4750565405 2540992255 1698817649 784832634 89061 ERX3511277 ERS3556005 ERA2100634 Computational Biology Unit|European Nucleotide Archive Computational Biology Unit 1 0.74003   0.5616   0.99855   0.03607   76   B   usable mapping rate illumina nextseq unknown other unknown bulk unknown unknown   Unknown 2019-07-03 Gastrula Embryo Undetermined Embryo Imprecise
9749 9749 ERR3489861 ERX3511276 ERS3556005 ERP116106 PRJEB33323 Deconstructing the individual steps of vertebrate translation initiation ena-STUDY-Computational Biology Unit-03-07-2019-10:11:34:314-422 Other In eukaryotes the number of ribosomes synthesizing a given protein depends on how many are recruited to its mRNA their success in navigating its five prime untranslated region UTR and whether they recognize its start codon. Initiation of translation is a rate limiting step in protein synthesis and key to gene expression control 1 but despite this centrality it remains poorly understood 2 3. Here we introduce ribosome complex profiling RCP seq to capture the transcriptome wide occupancy of scanning initiating elongating and terminating ribosome complexes in a higher eukaryote. We track scanning and elongating ribosomes across all five prime UTRs in zebrafish which enable us to assess the individual regulatory contributions from the three stages of initiation: ribosome recruitment scanning of the five prime UTR and recognition of the start codon. Our data sheds light on small subunit recruitment to mRNAs presenting evidence for the threading model and demonstrates that sequence features regulate this recruitment. We estimate the processivity of scanning ribosomes as they traverse the five prime UTR and show that the repressive effects of upstream open reading frames depend on the efficiency of both translation initiation and termination. Finally we determine the optimal initiation contexts by directly estimating the conversion of scanning to elongating ribosomes and demonstrate specific regulation of translation initiation at the endoplasmic reticulum. Our results open for the possibility of deconvoluting translation initiation into separate stages and provides the first view of global occupancy of ribosomal small subunits in a vertebrate. ENA FIRST PUBLIC:2020 03 27|ENA LAST UPDATE:2019 07 03     Shield 2 SAMEA5752546 Computational Biology Unit ENA FIRST PUBLIC:2020 03 27T17:04:59Z|ENA LAST UPDATE:2019 07 03T10:01:14Z|External Id:SAMEA5752546|INSDC center name:Computational Biology Unit|INSDC first public:2020 03 27T17:04:59Z|INSDC last update:2019 07 03T10:01:14Z|INSDC status:public|Submitter Id:9|common name:zebrafish|dev stage:Shield|sample name:9|scientific name:Danio rerio                 NextSeq 500 sequencing ena EXPERIMENT Computational Biology Unit 22 08 2019 11:57:43:424 13 Shield 2 SSU None RCP seq   OTHER TRANSCRIPTOMIC other SINGLE ILLUMINA NextSeq 500   ERP116106 NextSeq 500 sequencing ENA FIRST PUBLIC:2020 03 27|ENA LAST UPDATE:2019 08 22     8210103300.0 108027675.0 ena RUN Computational Biology Unit 22 08 2019 11:57:43:424 13 0:76 A:3300825043;C:2576709678;G:1707115198;T:625376255;N:77126 76       3300825043 2576709678 1707115198 625376255 77126 ERX3511276 ERS3556005 ERA2100634 Computational Biology Unit|European Nucleotide Archive Computational Biology Unit 1 0.2787   0.17583   0.99752   0.50171   76   B   usable mapping rate illumina nextseq unknown other unknown bulk unknown unknown   Unknown 2019-07-03 Gastrula Embryo Undetermined Embryo Imprecise
9750 9750 ERR3489860 ERX3511275 ERS3556004 ERP116106 PRJEB33323 Deconstructing the individual steps of vertebrate translation initiation ena-STUDY-Computational Biology Unit-03-07-2019-10:11:34:314-422 Other In eukaryotes the number of ribosomes synthesizing a given protein depends on how many are recruited to its mRNA their success in navigating its five prime untranslated region UTR and whether they recognize its start codon. Initiation of translation is a rate limiting step in protein synthesis and key to gene expression control 1 but despite this centrality it remains poorly understood 2 3. Here we introduce ribosome complex profiling RCP seq to capture the transcriptome wide occupancy of scanning initiating elongating and terminating ribosome complexes in a higher eukaryote. We track scanning and elongating ribosomes across all five prime UTRs in zebrafish which enable us to assess the individual regulatory contributions from the three stages of initiation: ribosome recruitment scanning of the five prime UTR and recognition of the start codon. Our data sheds light on small subunit recruitment to mRNAs presenting evidence for the threading model and demonstrates that sequence features regulate this recruitment. We estimate the processivity of scanning ribosomes as they traverse the five prime UTR and show that the repressive effects of upstream open reading frames depend on the efficiency of both translation initiation and termination. Finally we determine the optimal initiation contexts by directly estimating the conversion of scanning to elongating ribosomes and demonstrate specific regulation of translation initiation at the endoplasmic reticulum. Our results open for the possibility of deconvoluting translation initiation into separate stages and provides the first view of global occupancy of ribosomal small subunits in a vertebrate. ENA FIRST PUBLIC:2020 03 27|ENA LAST UPDATE:2019 07 03     Shield 1 SAMEA5752545 Computational Biology Unit ENA FIRST PUBLIC:2020 03 27T17:04:59Z|ENA LAST UPDATE:2019 07 03T10:01:14Z|External Id:SAMEA5752545|INSDC center name:Computational Biology Unit|INSDC first public:2020 03 27T17:04:59Z|INSDC last update:2019 07 03T10:01:14Z|INSDC status:public|Submitter Id:8|common name:zebrafish|dev stage:Shield|sample name:8|scientific name:Danio rerio                 NextSeq 500 sequencing ena EXPERIMENT Computational Biology Unit 22 08 2019 11:57:43:424 12 Shield 1 LSU None RCP seq   OTHER TRANSCRIPTOMIC other SINGLE ILLUMINA NextSeq 500   ERP116106 NextSeq 500 sequencing ENA FIRST PUBLIC:2020 03 27|ENA LAST UPDATE:2019 08 22     2437679936.0 32074736.0 ena RUN Computational Biology Unit 22 08 2019 11:57:43:424 12 0:76 A:764355184;C:710492003;G:664031460;T:298777374;N:23915 76       764355184 710492003 664031460 298777374 23915 ERX3511275 ERS3556004 ERA2100634 Computational Biology Unit|European Nucleotide Archive Computational Biology Unit 1 0.0406   0.02417   0.99908   0.61299   76   B   usable mapping rate illumina nextseq unknown other unknown bulk unknown unknown   Unknown 2019-07-03 Gastrula Embryo Undetermined Embryo Imprecise
9751 9751 ERR3489859 ERX3511274 ERS3556004 ERP116106 PRJEB33323 Deconstructing the individual steps of vertebrate translation initiation ena-STUDY-Computational Biology Unit-03-07-2019-10:11:34:314-422 Other In eukaryotes the number of ribosomes synthesizing a given protein depends on how many are recruited to its mRNA their success in navigating its five prime untranslated region UTR and whether they recognize its start codon. Initiation of translation is a rate limiting step in protein synthesis and key to gene expression control 1 but despite this centrality it remains poorly understood 2 3. Here we introduce ribosome complex profiling RCP seq to capture the transcriptome wide occupancy of scanning initiating elongating and terminating ribosome complexes in a higher eukaryote. We track scanning and elongating ribosomes across all five prime UTRs in zebrafish which enable us to assess the individual regulatory contributions from the three stages of initiation: ribosome recruitment scanning of the five prime UTR and recognition of the start codon. Our data sheds light on small subunit recruitment to mRNAs presenting evidence for the threading model and demonstrates that sequence features regulate this recruitment. We estimate the processivity of scanning ribosomes as they traverse the five prime UTR and show that the repressive effects of upstream open reading frames depend on the efficiency of both translation initiation and termination. Finally we determine the optimal initiation contexts by directly estimating the conversion of scanning to elongating ribosomes and demonstrate specific regulation of translation initiation at the endoplasmic reticulum. Our results open for the possibility of deconvoluting translation initiation into separate stages and provides the first view of global occupancy of ribosomal small subunits in a vertebrate. ENA FIRST PUBLIC:2020 03 27|ENA LAST UPDATE:2019 07 03     Shield 1 SAMEA5752545 Computational Biology Unit ENA FIRST PUBLIC:2020 03 27T17:04:59Z|ENA LAST UPDATE:2019 07 03T10:01:14Z|External Id:SAMEA5752545|INSDC center name:Computational Biology Unit|INSDC first public:2020 03 27T17:04:59Z|INSDC last update:2019 07 03T10:01:14Z|INSDC status:public|Submitter Id:8|common name:zebrafish|dev stage:Shield|sample name:8|scientific name:Danio rerio                 NextSeq 500 sequencing ena EXPERIMENT Computational Biology Unit 22 08 2019 11:57:43:424 11 Shield 1 SSU None RCP seq   OTHER TRANSCRIPTOMIC other SINGLE ILLUMINA NextSeq 500   ERP116106 NextSeq 500 sequencing ENA FIRST PUBLIC:2020 03 27|ENA LAST UPDATE:2019 08 22     3157243376.0 41542676.0 ena RUN Computational Biology Unit 22 08 2019 11:57:43:424 11 0:76 A:1443205052;C:715251024;G:633421305;T:365333650;N:32345 76       1443205052 715251024 633421305 365333650 32345 ERX3511274 ERS3556004 ERA2100634 Computational Biology Unit|European Nucleotide Archive Computational Biology Unit 1 0.47643   0.27944   0.99896   0.11464   76   B   usable mapping rate illumina nextseq unknown other unknown bulk unknown unknown   Unknown 2019-07-03 Gastrula Embryo Undetermined Embryo Imprecise
9752 9752 ERR3489858 ERX3511273 ERS3556001 ERP116106 PRJEB33323 Deconstructing the individual steps of vertebrate translation initiation ena-STUDY-Computational Biology Unit-03-07-2019-10:11:34:314-422 Other In eukaryotes the number of ribosomes synthesizing a given protein depends on how many are recruited to its mRNA their success in navigating its five prime untranslated region UTR and whether they recognize its start codon. Initiation of translation is a rate limiting step in protein synthesis and key to gene expression control 1 but despite this centrality it remains poorly understood 2 3. Here we introduce ribosome complex profiling RCP seq to capture the transcriptome wide occupancy of scanning initiating elongating and terminating ribosome complexes in a higher eukaryote. We track scanning and elongating ribosomes across all five prime UTRs in zebrafish which enable us to assess the individual regulatory contributions from the three stages of initiation: ribosome recruitment scanning of the five prime UTR and recognition of the start codon. Our data sheds light on small subunit recruitment to mRNAs presenting evidence for the threading model and demonstrates that sequence features regulate this recruitment. We estimate the processivity of scanning ribosomes as they traverse the five prime UTR and show that the repressive effects of upstream open reading frames depend on the efficiency of both translation initiation and termination. Finally we determine the optimal initiation contexts by directly estimating the conversion of scanning to elongating ribosomes and demonstrate specific regulation of translation initiation at the endoplasmic reticulum. Our results open for the possibility of deconvoluting translation initiation into separate stages and provides the first view of global occupancy of ribosomal small subunits in a vertebrate. ENA FIRST PUBLIC:2020 03 27|ENA LAST UPDATE:2019 07 03     Sphere 3 SAMEA5752542 Computational Biology Unit ENA FIRST PUBLIC:2020 03 27T17:04:59Z|ENA LAST UPDATE:2019 07 03T10:01:14Z|External Id:SAMEA5752542|INSDC center name:Computational Biology Unit|INSDC first public:2020 03 27T17:04:59Z|INSDC last update:2019 07 03T10:01:14Z|INSDC status:public|Submitter Id:5|common name:zebrafish|dev stage:Sphere|sample name:5|scientific name:Danio rerio                 NextSeq 500 sequencing ena EXPERIMENT Computational Biology Unit 22 08 2019 11:57:43:424 10 Sphere 3 LSU None RCP seq   OTHER TRANSCRIPTOMIC other SINGLE ILLUMINA NextSeq 500   ERP116106 NextSeq 500 sequencing ENA FIRST PUBLIC:2020 03 27|ENA LAST UPDATE:2019 08 22     2740392724.0 36057799.0 ena RUN Computational Biology Unit 22 08 2019 11:57:43:424 10 0:76 A:1790452280;C:354001675;G:431182140;T:164697730;N:58899 76       1790452280 354001675 431182140 164697730 58899 ERX3511273 ERS3556001 ERA2100634 Computational Biology Unit|European Nucleotide Archive Computational Biology Unit 1 0.87154   0.5323   0.99793   0.02983   76   B   usable mapping rate illumina nextseq unknown other unknown bulk unknown unknown   Unknown 2019-07-03 Blastula Embryo Undetermined Embryo Imprecise
9753 9753 ERR3489857 ERX3511272 ERS3556001 ERP116106 PRJEB33323 Deconstructing the individual steps of vertebrate translation initiation ena-STUDY-Computational Biology Unit-03-07-2019-10:11:34:314-422 Other In eukaryotes the number of ribosomes synthesizing a given protein depends on how many are recruited to its mRNA their success in navigating its five prime untranslated region UTR and whether they recognize its start codon. Initiation of translation is a rate limiting step in protein synthesis and key to gene expression control 1 but despite this centrality it remains poorly understood 2 3. Here we introduce ribosome complex profiling RCP seq to capture the transcriptome wide occupancy of scanning initiating elongating and terminating ribosome complexes in a higher eukaryote. We track scanning and elongating ribosomes across all five prime UTRs in zebrafish which enable us to assess the individual regulatory contributions from the three stages of initiation: ribosome recruitment scanning of the five prime UTR and recognition of the start codon. Our data sheds light on small subunit recruitment to mRNAs presenting evidence for the threading model and demonstrates that sequence features regulate this recruitment. We estimate the processivity of scanning ribosomes as they traverse the five prime UTR and show that the repressive effects of upstream open reading frames depend on the efficiency of both translation initiation and termination. Finally we determine the optimal initiation contexts by directly estimating the conversion of scanning to elongating ribosomes and demonstrate specific regulation of translation initiation at the endoplasmic reticulum. Our results open for the possibility of deconvoluting translation initiation into separate stages and provides the first view of global occupancy of ribosomal small subunits in a vertebrate. ENA FIRST PUBLIC:2020 03 27|ENA LAST UPDATE:2019 07 03     Sphere 3 SAMEA5752542 Computational Biology Unit ENA FIRST PUBLIC:2020 03 27T17:04:59Z|ENA LAST UPDATE:2019 07 03T10:01:14Z|External Id:SAMEA5752542|INSDC center name:Computational Biology Unit|INSDC first public:2020 03 27T17:04:59Z|INSDC last update:2019 07 03T10:01:14Z|INSDC status:public|Submitter Id:5|common name:zebrafish|dev stage:Sphere|sample name:5|scientific name:Danio rerio                 NextSeq 500 sequencing ena EXPERIMENT Computational Biology Unit 22 08 2019 11:57:43:424 9 Sphere 3 SSU None RCP seq   OTHER TRANSCRIPTOMIC other SINGLE ILLUMINA NextSeq 500   ERP116106 NextSeq 500 sequencing ENA FIRST PUBLIC:2020 03 27|ENA LAST UPDATE:2019 08 22     6277242192.0 82595292.0 ena RUN Computational Biology Unit 22 08 2019 11:57:43:424 9 0:76 A:3129446012;C:1311888416;G:1369035262;T:466745503;N:126999 76       3129446012 1311888416 1369035262 466745503 126999 ERX3511272 ERS3556001 ERA2100634 Computational Biology Unit|European Nucleotide Archive Computational Biology Unit 1 0.65637   0.35975   0.9936   0.24734   76   B   usable mapping rate illumina nextseq unknown other unknown bulk unknown unknown   Unknown 2019-07-03 Blastula Embryo Undetermined Embryo Imprecise
9754 9754 ERR3489856 ERX3511271 ERS3556000 ERP116106 PRJEB33323 Deconstructing the individual steps of vertebrate translation initiation ena-STUDY-Computational Biology Unit-03-07-2019-10:11:34:314-422 Other In eukaryotes the number of ribosomes synthesizing a given protein depends on how many are recruited to its mRNA their success in navigating its five prime untranslated region UTR and whether they recognize its start codon. Initiation of translation is a rate limiting step in protein synthesis and key to gene expression control 1 but despite this centrality it remains poorly understood 2 3. Here we introduce ribosome complex profiling RCP seq to capture the transcriptome wide occupancy of scanning initiating elongating and terminating ribosome complexes in a higher eukaryote. We track scanning and elongating ribosomes across all five prime UTRs in zebrafish which enable us to assess the individual regulatory contributions from the three stages of initiation: ribosome recruitment scanning of the five prime UTR and recognition of the start codon. Our data sheds light on small subunit recruitment to mRNAs presenting evidence for the threading model and demonstrates that sequence features regulate this recruitment. We estimate the processivity of scanning ribosomes as they traverse the five prime UTR and show that the repressive effects of upstream open reading frames depend on the efficiency of both translation initiation and termination. Finally we determine the optimal initiation contexts by directly estimating the conversion of scanning to elongating ribosomes and demonstrate specific regulation of translation initiation at the endoplasmic reticulum. Our results open for the possibility of deconvoluting translation initiation into separate stages and provides the first view of global occupancy of ribosomal small subunits in a vertebrate. ENA FIRST PUBLIC:2020 03 27|ENA LAST UPDATE:2019 07 03     Sphere 2 SAMEA5752541 Computational Biology Unit ENA FIRST PUBLIC:2020 03 27T17:04:59Z|ENA LAST UPDATE:2019 07 03T10:01:14Z|External Id:SAMEA5752541|INSDC center name:Computational Biology Unit|INSDC first public:2020 03 27T17:04:59Z|INSDC last update:2019 07 03T10:01:14Z|INSDC status:public|Submitter Id:4|common name:zebrafish|dev stage:Sphere|sample name:4|scientific name:Danio rerio                 NextSeq 500 sequencing ena EXPERIMENT Computational Biology Unit 22 08 2019 11:57:43:424 8 Sphere 2 LSU None RCP seq   OTHER TRANSCRIPTOMIC other SINGLE ILLUMINA NextSeq 500   ERP116106 NextSeq 500 sequencing ENA FIRST PUBLIC:2020 03 27|ENA LAST UPDATE:2019 08 24     6679993476.0 87894651.0 ena RUN Computational Biology Unit 22 08 2019 11:57:43:424 8 0:76 A:3113787833;C:1255798935;G:1750515950;T:559763997;N:126761 76       3113787833 1255798935 1750515950 559763997 126761 ERX3511271 ERS3556000 ERA2100634 Computational Biology Unit|European Nucleotide Archive Computational Biology Unit 1 0.66287   0.41208   0.99602   0.17083   76   B   usable mapping rate illumina nextseq unknown other unknown bulk unknown unknown   Unknown 2019-07-03 Blastula Embryo Undetermined Embryo Imprecise
9755 9755 ERR3489855 ERX3511270 ERS3556000 ERP116106 PRJEB33323 Deconstructing the individual steps of vertebrate translation initiation ena-STUDY-Computational Biology Unit-03-07-2019-10:11:34:314-422 Other In eukaryotes the number of ribosomes synthesizing a given protein depends on how many are recruited to its mRNA their success in navigating its five prime untranslated region UTR and whether they recognize its start codon. Initiation of translation is a rate limiting step in protein synthesis and key to gene expression control 1 but despite this centrality it remains poorly understood 2 3. Here we introduce ribosome complex profiling RCP seq to capture the transcriptome wide occupancy of scanning initiating elongating and terminating ribosome complexes in a higher eukaryote. We track scanning and elongating ribosomes across all five prime UTRs in zebrafish which enable us to assess the individual regulatory contributions from the three stages of initiation: ribosome recruitment scanning of the five prime UTR and recognition of the start codon. Our data sheds light on small subunit recruitment to mRNAs presenting evidence for the threading model and demonstrates that sequence features regulate this recruitment. We estimate the processivity of scanning ribosomes as they traverse the five prime UTR and show that the repressive effects of upstream open reading frames depend on the efficiency of both translation initiation and termination. Finally we determine the optimal initiation contexts by directly estimating the conversion of scanning to elongating ribosomes and demonstrate specific regulation of translation initiation at the endoplasmic reticulum. Our results open for the possibility of deconvoluting translation initiation into separate stages and provides the first view of global occupancy of ribosomal small subunits in a vertebrate. ENA FIRST PUBLIC:2020 03 27|ENA LAST UPDATE:2019 07 03     Sphere 2 SAMEA5752541 Computational Biology Unit ENA FIRST PUBLIC:2020 03 27T17:04:59Z|ENA LAST UPDATE:2019 07 03T10:01:14Z|External Id:SAMEA5752541|INSDC center name:Computational Biology Unit|INSDC first public:2020 03 27T17:04:59Z|INSDC last update:2019 07 03T10:01:14Z|INSDC status:public|Submitter Id:4|common name:zebrafish|dev stage:Sphere|sample name:4|scientific name:Danio rerio                 NextSeq 500 sequencing ena EXPERIMENT Computational Biology Unit 22 08 2019 11:57:43:423 7 Sphere 2 SSU None RCP seq   OTHER TRANSCRIPTOMIC other SINGLE ILLUMINA NextSeq 500   ERP116106 NextSeq 500 sequencing ENA FIRST PUBLIC:2020 03 27|ENA LAST UPDATE:2019 08 22     13238731764.0 174193839.0 ena RUN Computational Biology Unit 22 08 2019 11:57:43:424 7 0:76 A:7630016769;C:1835321568;G:2775223283;T:997916159;N:253985 76       7630016769 1835321568 2775223283 997916159 253985 ERX3511270 ERS3556000 ERA2100634 Computational Biology Unit|European Nucleotide Archive Computational Biology Unit 1 0.77269   0.45994   0.99683   0.04249   76   B   usable mapping rate illumina nextseq unknown other unknown bulk unknown unknown   Unknown 2019-07-03 Blastula Embryo Undetermined Embryo Imprecise
9756 9756 ERR3489854 ERX3511269 ERS3555999 ERP116106 PRJEB33323 Deconstructing the individual steps of vertebrate translation initiation ena-STUDY-Computational Biology Unit-03-07-2019-10:11:34:314-422 Other In eukaryotes the number of ribosomes synthesizing a given protein depends on how many are recruited to its mRNA their success in navigating its five prime untranslated region UTR and whether they recognize its start codon. Initiation of translation is a rate limiting step in protein synthesis and key to gene expression control 1 but despite this centrality it remains poorly understood 2 3. Here we introduce ribosome complex profiling RCP seq to capture the transcriptome wide occupancy of scanning initiating elongating and terminating ribosome complexes in a higher eukaryote. We track scanning and elongating ribosomes across all five prime UTRs in zebrafish which enable us to assess the individual regulatory contributions from the three stages of initiation: ribosome recruitment scanning of the five prime UTR and recognition of the start codon. Our data sheds light on small subunit recruitment to mRNAs presenting evidence for the threading model and demonstrates that sequence features regulate this recruitment. We estimate the processivity of scanning ribosomes as they traverse the five prime UTR and show that the repressive effects of upstream open reading frames depend on the efficiency of both translation initiation and termination. Finally we determine the optimal initiation contexts by directly estimating the conversion of scanning to elongating ribosomes and demonstrate specific regulation of translation initiation at the endoplasmic reticulum. Our results open for the possibility of deconvoluting translation initiation into separate stages and provides the first view of global occupancy of ribosomal small subunits in a vertebrate. ENA FIRST PUBLIC:2020 03 27|ENA LAST UPDATE:2019 07 03     Sphere 1 SAMEA5752540 Computational Biology Unit ENA FIRST PUBLIC:2020 03 27T17:04:59Z|ENA LAST UPDATE:2019 07 03T10:01:14Z|External Id:SAMEA5752540|INSDC center name:Computational Biology Unit|INSDC first public:2020 03 27T17:04:59Z|INSDC last update:2019 07 03T10:01:14Z|INSDC status:public|Submitter Id:3|common name:zebrafish|dev stage:Sphere|sample name:3|scientific name:Danio rerio                 NextSeq 500 sequencing ena EXPERIMENT Computational Biology Unit 22 08 2019 11:57:43:423 6 Sphere 1 LSU None RCP seq   OTHER TRANSCRIPTOMIC other SINGLE ILLUMINA NextSeq 500   ERP116106 NextSeq 500 sequencing ENA FIRST PUBLIC:2020 03 27|ENA LAST UPDATE:2019 08 24     3311799332.0 43576307.0 ena RUN Computational Biology Unit 22 08 2019 11:57:43:423 6 0:76 A:1182325939;C:888677953;G:922571204;T:318159754;N:64482 76       1182325939 888677953 922571204 318159754 64482 ERX3511269 ERS3555999 ERA2100634 Computational Biology Unit|European Nucleotide Archive Computational Biology Unit 1 0.57505   0.24459   0.99582   0.43365   76   B   usable mapping rate illumina nextseq unknown other unknown bulk unknown unknown   Unknown 2019-07-03 Blastula Embryo Undetermined Embryo Imprecise
9757 9757 ERR3489853 ERX3511268 ERS3555999 ERP116106 PRJEB33323 Deconstructing the individual steps of vertebrate translation initiation ena-STUDY-Computational Biology Unit-03-07-2019-10:11:34:314-422 Other In eukaryotes the number of ribosomes synthesizing a given protein depends on how many are recruited to its mRNA their success in navigating its five prime untranslated region UTR and whether they recognize its start codon. Initiation of translation is a rate limiting step in protein synthesis and key to gene expression control 1 but despite this centrality it remains poorly understood 2 3. Here we introduce ribosome complex profiling RCP seq to capture the transcriptome wide occupancy of scanning initiating elongating and terminating ribosome complexes in a higher eukaryote. We track scanning and elongating ribosomes across all five prime UTRs in zebrafish which enable us to assess the individual regulatory contributions from the three stages of initiation: ribosome recruitment scanning of the five prime UTR and recognition of the start codon. Our data sheds light on small subunit recruitment to mRNAs presenting evidence for the threading model and demonstrates that sequence features regulate this recruitment. We estimate the processivity of scanning ribosomes as they traverse the five prime UTR and show that the repressive effects of upstream open reading frames depend on the efficiency of both translation initiation and termination. Finally we determine the optimal initiation contexts by directly estimating the conversion of scanning to elongating ribosomes and demonstrate specific regulation of translation initiation at the endoplasmic reticulum. Our results open for the possibility of deconvoluting translation initiation into separate stages and provides the first view of global occupancy of ribosomal small subunits in a vertebrate. ENA FIRST PUBLIC:2020 03 27|ENA LAST UPDATE:2019 07 03     Sphere 1 SAMEA5752540 Computational Biology Unit ENA FIRST PUBLIC:2020 03 27T17:04:59Z|ENA LAST UPDATE:2019 07 03T10:01:14Z|External Id:SAMEA5752540|INSDC center name:Computational Biology Unit|INSDC first public:2020 03 27T17:04:59Z|INSDC last update:2019 07 03T10:01:14Z|INSDC status:public|Submitter Id:3|common name:zebrafish|dev stage:Sphere|sample name:3|scientific name:Danio rerio                 NextSeq 500 sequencing ena EXPERIMENT Computational Biology Unit 22 08 2019 11:57:43:423 5 Sphere 1 SSU None RCP seq   OTHER TRANSCRIPTOMIC other SINGLE ILLUMINA NextSeq 500   ERP116106 NextSeq 500 sequencing ENA FIRST PUBLIC:2020 03 27|ENA LAST UPDATE:2019 08 22     7403181508.0 97410283.0 ena RUN Computational Biology Unit 22 08 2019 11:57:43:423 5 0:76 A:4338227974;C:1233250165;G:1372229712;T:459322295;N:151362 76       4338227974 1233250165 1372229712 459322295 151362 ERX3511268 ERS3555999 ERA2100634 Computational Biology Unit|European Nucleotide Archive Computational Biology Unit 1 0.765   0.44747   0.99515   0.12467   76   B   usable mapping rate illumina nextseq unknown other unknown bulk unknown unknown   Unknown 2019-07-03 Blastula Embryo Undetermined Embryo Imprecise
9758 9758 ERR3489852 ERX3511267 ERS3555998 ERP116106 PRJEB33323 Deconstructing the individual steps of vertebrate translation initiation ena-STUDY-Computational Biology Unit-03-07-2019-10:11:34:314-422 Other In eukaryotes the number of ribosomes synthesizing a given protein depends on how many are recruited to its mRNA their success in navigating its five prime untranslated region UTR and whether they recognize its start codon. Initiation of translation is a rate limiting step in protein synthesis and key to gene expression control 1 but despite this centrality it remains poorly understood 2 3. Here we introduce ribosome complex profiling RCP seq to capture the transcriptome wide occupancy of scanning initiating elongating and terminating ribosome complexes in a higher eukaryote. We track scanning and elongating ribosomes across all five prime UTRs in zebrafish which enable us to assess the individual regulatory contributions from the three stages of initiation: ribosome recruitment scanning of the five prime UTR and recognition of the start codon. Our data sheds light on small subunit recruitment to mRNAs presenting evidence for the threading model and demonstrates that sequence features regulate this recruitment. We estimate the processivity of scanning ribosomes as they traverse the five prime UTR and show that the repressive effects of upstream open reading frames depend on the efficiency of both translation initiation and termination. Finally we determine the optimal initiation contexts by directly estimating the conversion of scanning to elongating ribosomes and demonstrate specific regulation of translation initiation at the endoplasmic reticulum. Our results open for the possibility of deconvoluting translation initiation into separate stages and provides the first view of global occupancy of ribosomal small subunits in a vertebrate. ENA FIRST PUBLIC:2020 03 27|ENA LAST UPDATE:2019 07 03     64 cell 2 SAMEA5752539 Computational Biology Unit ENA FIRST PUBLIC:2020 03 27T17:04:59Z|ENA LAST UPDATE:2019 07 03T10:01:14Z|External Id:SAMEA5752539|INSDC center name:Computational Biology Unit|INSDC first public:2020 03 27T17:04:59Z|INSDC last update:2019 07 03T10:01:14Z|INSDC status:public|Submitter Id:2|common name:zebrafish|dev stage:64 cell|sample name:2|scientific name:Danio rerio                 NextSeq 500 sequencing ena EXPERIMENT Computational Biology Unit 22 08 2019 11:57:43:423 4 64 cell 2 LSU None RCP seq   OTHER TRANSCRIPTOMIC other SINGLE ILLUMINA NextSeq 500   ERP116106 NextSeq 500 sequencing ENA FIRST PUBLIC:2020 03 27|ENA LAST UPDATE:2019 08 22     11722578884.0 154244459.0 ena RUN Computational Biology Unit 22 08 2019 11:57:43:423 4 0:76 A:8175036829;C:1396062240;G:1882846539;T:268587131;N:46145 76       8175036829 1396062240 1882846539 268587131 46145 ERX3511267 ERS3555998 ERA2100634 Computational Biology Unit|European Nucleotide Archive Computational Biology Unit 1 0.81133   0.41331   0.99823   0.03453   76   B   usable mapping rate illumina nextseq unknown other unknown bulk unknown unknown   Unknown 2019-07-03 Cleavage Embryo Undetermined Embryo Imprecise
9759 9759 ERR3489851 ERX3511266 ERS3555998 ERP116106 PRJEB33323 Deconstructing the individual steps of vertebrate translation initiation ena-STUDY-Computational Biology Unit-03-07-2019-10:11:34:314-422 Other In eukaryotes the number of ribosomes synthesizing a given protein depends on how many are recruited to its mRNA their success in navigating its five prime untranslated region UTR and whether they recognize its start codon. Initiation of translation is a rate limiting step in protein synthesis and key to gene expression control 1 but despite this centrality it remains poorly understood 2 3. Here we introduce ribosome complex profiling RCP seq to capture the transcriptome wide occupancy of scanning initiating elongating and terminating ribosome complexes in a higher eukaryote. We track scanning and elongating ribosomes across all five prime UTRs in zebrafish which enable us to assess the individual regulatory contributions from the three stages of initiation: ribosome recruitment scanning of the five prime UTR and recognition of the start codon. Our data sheds light on small subunit recruitment to mRNAs presenting evidence for the threading model and demonstrates that sequence features regulate this recruitment. We estimate the processivity of scanning ribosomes as they traverse the five prime UTR and show that the repressive effects of upstream open reading frames depend on the efficiency of both translation initiation and termination. Finally we determine the optimal initiation contexts by directly estimating the conversion of scanning to elongating ribosomes and demonstrate specific regulation of translation initiation at the endoplasmic reticulum. Our results open for the possibility of deconvoluting translation initiation into separate stages and provides the first view of global occupancy of ribosomal small subunits in a vertebrate. ENA FIRST PUBLIC:2020 03 27|ENA LAST UPDATE:2019 07 03     64 cell 2 SAMEA5752539 Computational Biology Unit ENA FIRST PUBLIC:2020 03 27T17:04:59Z|ENA LAST UPDATE:2019 07 03T10:01:14Z|External Id:SAMEA5752539|INSDC center name:Computational Biology Unit|INSDC first public:2020 03 27T17:04:59Z|INSDC last update:2019 07 03T10:01:14Z|INSDC status:public|Submitter Id:2|common name:zebrafish|dev stage:64 cell|sample name:2|scientific name:Danio rerio                 NextSeq 500 sequencing ena EXPERIMENT Computational Biology Unit 22 08 2019 11:57:43:423 3 64 cell 2 SSU None RCP seq   OTHER TRANSCRIPTOMIC other SINGLE ILLUMINA NextSeq 500   ERP116106 NextSeq 500 sequencing ENA FIRST PUBLIC:2020 03 27|ENA LAST UPDATE:2019 08 22     9525478088.0 125335238.0 ena RUN Computational Biology Unit 22 08 2019 11:57:43:423 3 0:76 A:4813606784;C:2048902072;G:2140220088;T:522714000;N:35144 76       4813606784 2048902072 2140220088 522714000 35144 ERX3511266 ERS3555998 ERA2100634 Computational Biology Unit|European Nucleotide Archive Computational Biology Unit 1 0.59747   0.26784   0.996   0.21193   76   B   usable mapping rate illumina nextseq unknown other unknown bulk unknown unknown   Unknown 2019-07-03 Cleavage Embryo Undetermined Embryo Imprecise
9760 9760 ERR3489850 ERX3511265 ERS3555997 ERP116106 PRJEB33323 Deconstructing the individual steps of vertebrate translation initiation ena-STUDY-Computational Biology Unit-03-07-2019-10:11:34:314-422 Other In eukaryotes the number of ribosomes synthesizing a given protein depends on how many are recruited to its mRNA their success in navigating its five prime untranslated region UTR and whether they recognize its start codon. Initiation of translation is a rate limiting step in protein synthesis and key to gene expression control 1 but despite this centrality it remains poorly understood 2 3. Here we introduce ribosome complex profiling RCP seq to capture the transcriptome wide occupancy of scanning initiating elongating and terminating ribosome complexes in a higher eukaryote. We track scanning and elongating ribosomes across all five prime UTRs in zebrafish which enable us to assess the individual regulatory contributions from the three stages of initiation: ribosome recruitment scanning of the five prime UTR and recognition of the start codon. Our data sheds light on small subunit recruitment to mRNAs presenting evidence for the threading model and demonstrates that sequence features regulate this recruitment. We estimate the processivity of scanning ribosomes as they traverse the five prime UTR and show that the repressive effects of upstream open reading frames depend on the efficiency of both translation initiation and termination. Finally we determine the optimal initiation contexts by directly estimating the conversion of scanning to elongating ribosomes and demonstrate specific regulation of translation initiation at the endoplasmic reticulum. Our results open for the possibility of deconvoluting translation initiation into separate stages and provides the first view of global occupancy of ribosomal small subunits in a vertebrate. ENA FIRST PUBLIC:2020 03 27|ENA LAST UPDATE:2019 07 03     64 cell 1 SAMEA5752538 Computational Biology Unit ENA FIRST PUBLIC:2020 03 27T17:04:59Z|ENA LAST UPDATE:2019 07 03T10:01:14Z|External Id:SAMEA5752538|INSDC center name:Computational Biology Unit|INSDC first public:2020 03 27T17:04:59Z|INSDC last update:2019 07 03T10:01:14Z|INSDC status:public|Submitter Id:1|common name:zebrafish|dev stage:64 cell|sample name:1|scientific name:Danio rerio                 NextSeq 500 sequencing ena EXPERIMENT Computational Biology Unit 22 08 2019 11:57:43:423 2 64 cell 1 LSU None RCP seq   OTHER TRANSCRIPTOMIC other SINGLE ILLUMINA NextSeq 500   ERP116106 NextSeq 500 sequencing ENA FIRST PUBLIC:2020 03 27|ENA LAST UPDATE:2019 08 22     8544157652.0 112423127.0 ena RUN Computational Biology Unit 22 08 2019 11:57:43:423 2 0:76 A:2633354802;C:2582123846;G:2505786164;T:822713754;N:179086 76       2633354802 2582123846 2505786164 822713754 179086 ERX3511265 ERS3555997 ERA2100634 Computational Biology Unit|European Nucleotide Archive Computational Biology Unit 1 0.34707   0.02129   0.99797   0.62478   76   B   usable mapping rate illumina nextseq unknown other unknown bulk unknown unknown   Unknown 2019-07-03 Cleavage Embryo Undetermined Embryo Imprecise
9761 9761 ERR3489849 ERX3511264 ERS3555997 ERP116106 PRJEB33323 Deconstructing the individual steps of vertebrate translation initiation ena-STUDY-Computational Biology Unit-03-07-2019-10:11:34:314-422 Other In eukaryotes the number of ribosomes synthesizing a given protein depends on how many are recruited to its mRNA their success in navigating its five prime untranslated region UTR and whether they recognize its start codon. Initiation of translation is a rate limiting step in protein synthesis and key to gene expression control 1 but despite this centrality it remains poorly understood 2 3. Here we introduce ribosome complex profiling RCP seq to capture the transcriptome wide occupancy of scanning initiating elongating and terminating ribosome complexes in a higher eukaryote. We track scanning and elongating ribosomes across all five prime UTRs in zebrafish which enable us to assess the individual regulatory contributions from the three stages of initiation: ribosome recruitment scanning of the five prime UTR and recognition of the start codon. Our data sheds light on small subunit recruitment to mRNAs presenting evidence for the threading model and demonstrates that sequence features regulate this recruitment. We estimate the processivity of scanning ribosomes as they traverse the five prime UTR and show that the repressive effects of upstream open reading frames depend on the efficiency of both translation initiation and termination. Finally we determine the optimal initiation contexts by directly estimating the conversion of scanning to elongating ribosomes and demonstrate specific regulation of translation initiation at the endoplasmic reticulum. Our results open for the possibility of deconvoluting translation initiation into separate stages and provides the first view of global occupancy of ribosomal small subunits in a vertebrate. ENA FIRST PUBLIC:2020 03 27|ENA LAST UPDATE:2019 07 03     64 cell 1 SAMEA5752538 Computational Biology Unit ENA FIRST PUBLIC:2020 03 27T17:04:59Z|ENA LAST UPDATE:2019 07 03T10:01:14Z|External Id:SAMEA5752538|INSDC center name:Computational Biology Unit|INSDC first public:2020 03 27T17:04:59Z|INSDC last update:2019 07 03T10:01:14Z|INSDC status:public|Submitter Id:1|common name:zebrafish|dev stage:64 cell|sample name:1|scientific name:Danio rerio                 NextSeq 500 sequencing ena EXPERIMENT Computational Biology Unit 22 08 2019 11:57:43:423 1 64 cell 1 SSU None RCP seq   OTHER TRANSCRIPTOMIC other SINGLE ILLUMINA NextSeq 500   ERP116106 NextSeq 500 sequencing ENA FIRST PUBLIC:2020 03 27|ENA LAST UPDATE:2019 08 22 run7_64_cell_SSU_12_13_14.fastq.gz fastq 10139466432.0 133414032.0 ena RUN Computational Biology Unit 22 08 2019 11:57:43:423 1 0:76 1:0 A:4628193785;C:2494821868;G:2485640988;T:530605907;N:203884 76 0     4628193785 2494821868 2485640988 530605907 203884 ERX3511264 ERS3555997 ERA2100634 Computational Biology Unit|European Nucleotide Archive Computational Biology Unit 1 0.06893   0.02559   0.99766   0.90567   76   B   usable mapping rate illumina nextseq unknown other unknown bulk unknown unknown   Unknown 2019-07-03 Cleavage Embryo Undetermined Embryo Imprecise
9762 9762 ERR3413870 ERX3437516 ERS3555997 ERP116106 PRJEB33323 Deconstructing the individual steps of vertebrate translation initiation ena-STUDY-Computational Biology Unit-03-07-2019-10:11:34:314-422 Other In eukaryotes the number of ribosomes synthesizing a given protein depends on how many are recruited to its mRNA their success in navigating its five prime untranslated region UTR and whether they recognize its start codon. Initiation of translation is a rate limiting step in protein synthesis and key to gene expression control 1 but despite this centrality it remains poorly understood 2 3. Here we introduce ribosome complex profiling RCP seq to capture the transcriptome wide occupancy of scanning initiating elongating and terminating ribosome complexes in a higher eukaryote. We track scanning and elongating ribosomes across all five prime UTRs in zebrafish which enable us to assess the individual regulatory contributions from the three stages of initiation: ribosome recruitment scanning of the five prime UTR and recognition of the start codon. Our data sheds light on small subunit recruitment to mRNAs presenting evidence for the threading model and demonstrates that sequence features regulate this recruitment. We estimate the processivity of scanning ribosomes as they traverse the five prime UTR and show that the repressive effects of upstream open reading frames depend on the efficiency of both translation initiation and termination. Finally we determine the optimal initiation contexts by directly estimating the conversion of scanning to elongating ribosomes and demonstrate specific regulation of translation initiation at the endoplasmic reticulum. Our results open for the possibility of deconvoluting translation initiation into separate stages and provides the first view of global occupancy of ribosomal small subunits in a vertebrate. ENA FIRST PUBLIC:2020 03 27|ENA LAST UPDATE:2019 07 03     64 cell 1 SAMEA5752538 Computational Biology Unit ENA FIRST PUBLIC:2020 03 27T17:04:59Z|ENA LAST UPDATE:2019 07 03T10:01:14Z|External Id:SAMEA5752538|INSDC center name:Computational Biology Unit|INSDC first public:2020 03 27T17:04:59Z|INSDC last update:2019 07 03T10:01:14Z|INSDC status:public|Submitter Id:1|common name:zebrafish|dev stage:64 cell|sample name:1|scientific name:Danio rerio                 NextSeq 500 sequencing ena EXPERIMENT Computational Biology Unit 03 07 2019 14:45:09:705 2 64 cell 1 LSU None RCP seq   OTHER TRANSCRIPTOMIC other SINGLE ILLUMINA NextSeq 500   ERP116106 NextSeq 500 sequencing ENA FIRST PUBLIC:2020 03 27|ENA LAST UPDATE:2019 07 03     8544157652.0 112423127.0 ena RUN Computational Biology Unit 03 07 2019 14:45:09:705 2 0:76 A:2633354802;C:2582123846;G:2505786164;T:822713754;N:179086 76       2633354802 2582123846 2505786164 822713754 179086 ERX3437516 ERS3555997 ERA2028987 Computational Biology Unit|European Nucleotide Archive Computational Biology Unit 1 0.34696   0.02086   0.99795   0.66261   76   B   usable mapping rate illumina nextseq unknown other unknown bulk unknown unknown   Unknown 2019-07-03 Cleavage Embryo Undetermined Embryo Imprecise
9763 9763 ERR3413869 ERX3437515 ERS3555997 ERP116106 PRJEB33323 Deconstructing the individual steps of vertebrate translation initiation ena-STUDY-Computational Biology Unit-03-07-2019-10:11:34:314-422 Other In eukaryotes the number of ribosomes synthesizing a given protein depends on how many are recruited to its mRNA their success in navigating its five prime untranslated region UTR and whether they recognize its start codon. Initiation of translation is a rate limiting step in protein synthesis and key to gene expression control 1 but despite this centrality it remains poorly understood 2 3. Here we introduce ribosome complex profiling RCP seq to capture the transcriptome wide occupancy of scanning initiating elongating and terminating ribosome complexes in a higher eukaryote. We track scanning and elongating ribosomes across all five prime UTRs in zebrafish which enable us to assess the individual regulatory contributions from the three stages of initiation: ribosome recruitment scanning of the five prime UTR and recognition of the start codon. Our data sheds light on small subunit recruitment to mRNAs presenting evidence for the threading model and demonstrates that sequence features regulate this recruitment. We estimate the processivity of scanning ribosomes as they traverse the five prime UTR and show that the repressive effects of upstream open reading frames depend on the efficiency of both translation initiation and termination. Finally we determine the optimal initiation contexts by directly estimating the conversion of scanning to elongating ribosomes and demonstrate specific regulation of translation initiation at the endoplasmic reticulum. Our results open for the possibility of deconvoluting translation initiation into separate stages and provides the first view of global occupancy of ribosomal small subunits in a vertebrate. ENA FIRST PUBLIC:2020 03 27|ENA LAST UPDATE:2019 07 03     64 cell 1 SAMEA5752538 Computational Biology Unit ENA FIRST PUBLIC:2020 03 27T17:04:59Z|ENA LAST UPDATE:2019 07 03T10:01:14Z|External Id:SAMEA5752538|INSDC center name:Computational Biology Unit|INSDC first public:2020 03 27T17:04:59Z|INSDC last update:2019 07 03T10:01:14Z|INSDC status:public|Submitter Id:1|common name:zebrafish|dev stage:64 cell|sample name:1|scientific name:Danio rerio                 NextSeq 500 sequencing ena EXPERIMENT Computational Biology Unit 03 07 2019 14:45:09:705 1 64 cell 1 SSU None RCP seq   OTHER TRANSCRIPTOMIC other SINGLE ILLUMINA NextSeq 500   ERP116106 NextSeq 500 sequencing ENA FIRST PUBLIC:2020 03 27|ENA LAST UPDATE:2019 07 03     10139466432.0 133414032.0 ena RUN Computational Biology Unit 03 07 2019 14:45:09:705 1 0:76 A:4628193785;C:2494821868;G:2485640988;T:530605907;N:203884 76       4628193785 2494821868 2485640988 530605907 203884 ERX3437515 ERS3555997 ERA2028987 Computational Biology Unit|European Nucleotide Archive Computational Biology Unit 1 0.06884   0.02526   0.99762   0.89856   76   B   usable mapping rate illumina nextseq unknown other unknown bulk unknown unknown   Unknown 2019-07-03 Cleavage Embryo Undetermined Embryo Imprecise
25182 25182 SRR25670729 SRX21396042 SRS18636200 SRP455680 PRJNA1006406 Control of polyA tail length and translation in vertebrate oocytes and early embryos GSE241107 Other During oocyte maturation and early embryonic development polyA tail lengths strongly influence mRNA translation. However how tail lengths are controlled at different developmental stages has been unclear. Here we performed tail length and translational profiling of mRNA reporter libraries each with > 10 million three prime UTR sequence variants in frog oocytes and embryos and fish embryos. These analyses revealed that the UUUUA motif specifies cytoplasmic polyadenylation and identified diverse context features that modulate the activity of this 5 mer. Additional sequence motifs drive stage specific deadenylation in embryos and UUUUA and C rich motifs drive tail length independent translational repression in oocytes. A neural network model accurately predicts tail length change during oocyte maturation in frogs mice and humans. Analyses of human sequence variants showed that those predicted to disrupt tail length control have been under negative selection implying that our insights into control of polyA tail length and translation have implications for human health and fertility. Overall design: Sythetic mRNA reporter libraries with random three prime UTR sequences under four different sequence contexts were injected into frog oocytes and embryos and fish embryos. PolyA tail lengths were measured to at different developmental stages to examine sequence motifs that caused tail length changes. At the same time polyA tail lengths of endogenous mRNAs from frog oocytes and embryos fish embryos and mouse oocytes were measured to investiage how their tail lengths were controlled. Please note that sample titles have been updated on Jan 6 2024.       Fish embryo mRNA germ ring PAL seq v4 GSM7716871   source name:embryo|tissue:embryo|treatment:N1|geo loc name:missing|collection date:missing Fish embryo mRNA germ ring PAL seq v4 For gene specific tail seq of mRNA reporters data were processed with a custom script available at https://github.com/coffeebond/MPRA tail seq. For PAL seq v3 or v4 reads were trimmed with cutadapt v3.7 with the parameters “ m 15 quality base=64 q 20 20 match read wildcards e 0.05 a NNNNATCTCGTATGCCGTCTTCTGCTTG O 7”. The trimmed reads were mapped using STAR v2.7.1a to the reference database containing the genomic sequences of the organism from which the mRNAs were obtained the genomic sequences of humans or fish depending on which spike in RNAs were used and the sequences of the polyA standards generated previously with the parameters “ runThreadN 16 runMode alignReads outFilterMultimapNmax 1 outReadsUnmapped Fastx outFilterType BySJout outSAMattributes All outSAMtype BAM Unsorted SortedByCoordinate”. Uniquely mapped read were furhter processed with a custom script available at https://github.com/coffeebond/PAL seq. Assembly: Homo sapiens: GRCh38.p7 primary assembly. Mus muculus: GRCh38.p4 primary assembly. Xenopus laevis: v10.1 assembly. Danio rerio: GRCz11 assembly. Supplementary files format and content: For gene specific tail seq of mRNA reporters tab delimited text files with columns indicating 1 sequence of the variable region; 2 median tail length; 3 number of reads; 4–7 tail lengths at quantile 10 25 75 and 90. Supplementary files format and content: For PAL seq v3 or v4 tab delimited files with columns indicating 1 gene ID or polyA site ID; 2 read cluster ID; 3 tail length Library strategy: PAL seq v4 embryo   Frog oocytes and embryos were lysed in ice cold buffer RL 20 mM HEPES pH 7.5 100 mM KCl 5 mM MgCl2 1% [v/v] Triton X 100 100 µg/ml cycloheximide cOmplete protease inhibitor cocktail [1 tablet per 10 ml buffer] and 200 units/ml SUPERase•In in a volume of 10 µl per oocyte/embryo by vigorous shaking and pipetting. Lysates were cleared by centrifugation at 5000 g at 4°C for 10 min. The supernatant was transfered to a new tube and mixed with the Tri reagent for RNA isolation. Fish embryos were de chorionated by incubation with 2 mg/ml pronase in E3 medium for 4 min. post removing all E3 medium Tri Reagent was added to the embryos for RNA isolation. Mouse GV oocytes were collected in 37°C MEM in the presence of milrinone to prevent maturation. Cumulus cells were removed from cumulus oocyte complexes by repeated aspiration through a glass pipette. GV oocytes were then collected in TRI reagent for RNA isolation. Mouse MII oocytes were harvested from the oviducts of hCG induced mice 16 hr denuded with 3 mg/ml hyaluronidase for 2 min washed and then collected in Tri reagent for RNA isolation. For gene specific tail seq of reporter libraries total RNA with reporter mRNA libraries was ligated to a pre adenylated 3ʹ adapter directly in most cases but for the N60 library injected into fish embryos and frog oocytes the N37 PAS N17 library injected into fish embryos and frog oocytes and the CPEmos N60 and N60LC PASmos libraries injected into frog oocytes reporter library mRNAs were enriched by anti sense oligo capture with biotinylated oligos. RNA isolated from the oocyte or embryo lysate was mixed with 8 pmol KXSH009 8 pmol KXSH010 and 2x SSC 0.3 M NaCl 30 mM sodium citrate pH 7.0 in a total of 50 µl. The RNA and oligos were annealed by incubation at 70°C for 5 min and then slowly cooling to 23°C at 0.1°C/sec. The annealed mixture was combined with 40 µl MyOne Streptavidin C1 beads Thermo Fisher 65002 and incubated for 20 min at 23°C on a thermal mixer shaking with 15 sec on and 1 min 45 sec off. The superna…   tissue:embryo|treatment:N1 GSM7716871 GSM7716871: Fish embryo mRNA germ ring PAL seq v4; Danio rerio; OTHER GSM7716871 r1 GSM7716871 1 Frog oocytes and embryos were lysed in ice cold buffer RL 20 mM HEPES pH 7.5 100 mM KCl 5 mM MgCl2 1% [v/v] Triton X 100 100 µg/ml cycloheximide cOmplete protease inhibitor cocktail [1 tablet per 10 ml buffer] and 200 units/ml SUPERase•In in a volume of 10 µl per oocyte/embryo by vigorous shaking and pipetting. Lysates were cleared by centrifugation at 5000 g at 4°C for 10 min. The supernatant was transfered to a new tube and mixed with the Tri reagent for RNA isolation. Fish embryos were de chorionated by incubation with 2 mg/ml pronase in E3 medium for 4 min. post removing all E3 medium Tri Reagent was added to the embryos for RNA isolation. Mouse GV oocytes were collected in 37°C MEM in the presence of milrinone to prevent maturation. Cumulus cells were removed from cumulus oocyte complexes by repeated aspiration through a glass pipette. GV oocytes were then collected in TRI reagent for RNA isolation. Mouse MII oocytes were harvested from the oviducts of hCG induced mice 16 hr denuded with 3 mg/ml hyaluronidase for 2 min washed and then collected in Tri reagent for RNA isolation. For gene specific tail seq of reporter libraries total RNA with reporter mRNA libraries was ligated to a pre adenylated 3ʹ adapter directly in most cases but for the N60 library injected into fish embryos and frog oocytes the N37 PAS N17 library injected into fish embryos and frog oocytes and the CPEmos N60 and N60LC PASmos libraries injected into frog oocytes reporter library mRNAs were enriched by anti sense oligo capture with biotinylated oligos. RNA isolated from the oocyte or embryo lysate was mixed with 8 pmol KXSH009 8 pmol KXSH010 and 2x SSC 0.3 M NaCl 30 mM sodium citrate pH 7.0 in a total of 50 µl. The RNA and oligos were annealed by incubation at 70°C for 5 min and then slowly cooling to 23°C at 0.1°C/sec. The annealed mixture was combined with 40 µl MyOne Streptavidin C1 beads Thermo Fisher 65002 and incubated for 20 min at 23°C on a thermal mixer shaking with 15 sec on and 1 min 45 sec off. The superna…   OTHER TRANSCRIPTOMIC other PAIRED ILLUMINA Illumina HiSeq 2500   SRP455680     Fish_embryo_mRNA_germ_ring_PAL_seq_v4_rep1_raw_read2.fastq.gz Fish_embryo_mRNA_germ_ring_PAL_seq_v4_rep1_raw_read1.fastq.gz fastq fastq 2834842912.0 9234016.0 GSM7716871 r1 0:52 1:255 A:725811118;C:702410592;G:747583409;T:651018348;N:8019445 52 255     725811118 702410592 747583409 651018348 8019445 SRX21396042 SRS18636200 SRA1694849 Whitehead Institute Whitehead Institute 2 0.00023 0.30494 8e-05 0.01793 0.99967 0.99971 0.5 1.0 52 255 T B mate1 technical by mapping diff illumina hiseq_era unknown poly_a unknown bulk bulk bulk   United States 2023-08-17 Multi-stage Embryo Embryo Imprecise All anatomical structures
25183 25183 SRR25670730 SRX21396042 SRS18636200 SRP455680 PRJNA1006406 Control of polyA tail length and translation in vertebrate oocytes and early embryos GSE241107 Other During oocyte maturation and early embryonic development polyA tail lengths strongly influence mRNA translation. However how tail lengths are controlled at different developmental stages has been unclear. Here we performed tail length and translational profiling of mRNA reporter libraries each with > 10 million three prime UTR sequence variants in frog oocytes and embryos and fish embryos. These analyses revealed that the UUUUA motif specifies cytoplasmic polyadenylation and identified diverse context features that modulate the activity of this 5 mer. Additional sequence motifs drive stage specific deadenylation in embryos and UUUUA and C rich motifs drive tail length independent translational repression in oocytes. A neural network model accurately predicts tail length change during oocyte maturation in frogs mice and humans. Analyses of human sequence variants showed that those predicted to disrupt tail length control have been under negative selection implying that our insights into control of polyA tail length and translation have implications for human health and fertility. Overall design: Sythetic mRNA reporter libraries with random three prime UTR sequences under four different sequence contexts were injected into frog oocytes and embryos and fish embryos. PolyA tail lengths were measured to at different developmental stages to examine sequence motifs that caused tail length changes. At the same time polyA tail lengths of endogenous mRNAs from frog oocytes and embryos fish embryos and mouse oocytes were measured to investiage how their tail lengths were controlled. Please note that sample titles have been updated on Jan 6 2024.       Fish embryo mRNA germ ring PAL seq v4 GSM7716871   source name:embryo|tissue:embryo|treatment:N1|geo loc name:missing|collection date:missing Fish embryo mRNA germ ring PAL seq v4 For gene specific tail seq of mRNA reporters data were processed with a custom script available at https://github.com/coffeebond/MPRA tail seq. For PAL seq v3 or v4 reads were trimmed with cutadapt v3.7 with the parameters “ m 15 quality base=64 q 20 20 match read wildcards e 0.05 a NNNNATCTCGTATGCCGTCTTCTGCTTG O 7”. The trimmed reads were mapped using STAR v2.7.1a to the reference database containing the genomic sequences of the organism from which the mRNAs were obtained the genomic sequences of humans or fish depending on which spike in RNAs were used and the sequences of the polyA standards generated previously with the parameters “ runThreadN 16 runMode alignReads outFilterMultimapNmax 1 outReadsUnmapped Fastx outFilterType BySJout outSAMattributes All outSAMtype BAM Unsorted SortedByCoordinate”. Uniquely mapped read were furhter processed with a custom script available at https://github.com/coffeebond/PAL seq. Assembly: Homo sapiens: GRCh38.p7 primary assembly. Mus muculus: GRCh38.p4 primary assembly. Xenopus laevis: v10.1 assembly. Danio rerio: GRCz11 assembly. Supplementary files format and content: For gene specific tail seq of mRNA reporters tab delimited text files with columns indicating 1 sequence of the variable region; 2 median tail length; 3 number of reads; 4–7 tail lengths at quantile 10 25 75 and 90. Supplementary files format and content: For PAL seq v3 or v4 tab delimited files with columns indicating 1 gene ID or polyA site ID; 2 read cluster ID; 3 tail length Library strategy: PAL seq v4 embryo   Frog oocytes and embryos were lysed in ice cold buffer RL 20 mM HEPES pH 7.5 100 mM KCl 5 mM MgCl2 1% [v/v] Triton X 100 100 µg/ml cycloheximide cOmplete protease inhibitor cocktail [1 tablet per 10 ml buffer] and 200 units/ml SUPERase•In in a volume of 10 µl per oocyte/embryo by vigorous shaking and pipetting. Lysates were cleared by centrifugation at 5000 g at 4°C for 10 min. The supernatant was transfered to a new tube and mixed with the Tri reagent for RNA isolation. Fish embryos were de chorionated by incubation with 2 mg/ml pronase in E3 medium for 4 min. post removing all E3 medium Tri Reagent was added to the embryos for RNA isolation. Mouse GV oocytes were collected in 37°C MEM in the presence of milrinone to prevent maturation. Cumulus cells were removed from cumulus oocyte complexes by repeated aspiration through a glass pipette. GV oocytes were then collected in TRI reagent for RNA isolation. Mouse MII oocytes were harvested from the oviducts of hCG induced mice 16 hr denuded with 3 mg/ml hyaluronidase for 2 min washed and then collected in Tri reagent for RNA isolation. For gene specific tail seq of reporter libraries total RNA with reporter mRNA libraries was ligated to a pre adenylated 3ʹ adapter directly in most cases but for the N60 library injected into fish embryos and frog oocytes the N37 PAS N17 library injected into fish embryos and frog oocytes and the CPEmos N60 and N60LC PASmos libraries injected into frog oocytes reporter library mRNAs were enriched by anti sense oligo capture with biotinylated oligos. RNA isolated from the oocyte or embryo lysate was mixed with 8 pmol KXSH009 8 pmol KXSH010 and 2x SSC 0.3 M NaCl 30 mM sodium citrate pH 7.0 in a total of 50 µl. The RNA and oligos were annealed by incubation at 70°C for 5 min and then slowly cooling to 23°C at 0.1°C/sec. The annealed mixture was combined with 40 µl MyOne Streptavidin C1 beads Thermo Fisher 65002 and incubated for 20 min at 23°C on a thermal mixer shaking with 15 sec on and 1 min 45 sec off. The superna…   tissue:embryo|treatment:N1 GSM7716871 GSM7716871: Fish embryo mRNA germ ring PAL seq v4; Danio rerio; OTHER GSM7716871 r1 GSM7716871 1 Frog oocytes and embryos were lysed in ice cold buffer RL 20 mM HEPES pH 7.5 100 mM KCl 5 mM MgCl2 1% [v/v] Triton X 100 100 µg/ml cycloheximide cOmplete protease inhibitor cocktail [1 tablet per 10 ml buffer] and 200 units/ml SUPERase•In in a volume of 10 µl per oocyte/embryo by vigorous shaking and pipetting. Lysates were cleared by centrifugation at 5000 g at 4°C for 10 min. The supernatant was transfered to a new tube and mixed with the Tri reagent for RNA isolation. Fish embryos were de chorionated by incubation with 2 mg/ml pronase in E3 medium for 4 min. post removing all E3 medium Tri Reagent was added to the embryos for RNA isolation. Mouse GV oocytes were collected in 37°C MEM in the presence of milrinone to prevent maturation. Cumulus cells were removed from cumulus oocyte complexes by repeated aspiration through a glass pipette. GV oocytes were then collected in TRI reagent for RNA isolation. Mouse MII oocytes were harvested from the oviducts of hCG induced mice 16 hr denuded with 3 mg/ml hyaluronidase for 2 min washed and then collected in Tri reagent for RNA isolation. For gene specific tail seq of reporter libraries total RNA with reporter mRNA libraries was ligated to a pre adenylated 3ʹ adapter directly in most cases but for the N60 library injected into fish embryos and frog oocytes the N37 PAS N17 library injected into fish embryos and frog oocytes and the CPEmos N60 and N60LC PASmos libraries injected into frog oocytes reporter library mRNAs were enriched by anti sense oligo capture with biotinylated oligos. RNA isolated from the oocyte or embryo lysate was mixed with 8 pmol KXSH009 8 pmol KXSH010 and 2x SSC 0.3 M NaCl 30 mM sodium citrate pH 7.0 in a total of 50 µl. The RNA and oligos were annealed by incubation at 70°C for 5 min and then slowly cooling to 23°C at 0.1°C/sec. The annealed mixture was combined with 40 µl MyOne Streptavidin C1 beads Thermo Fisher 65002 and incubated for 20 min at 23°C on a thermal mixer shaking with 15 sec on and 1 min 45 sec off. The superna…   OTHER TRANSCRIPTOMIC other PAIRED ILLUMINA Illumina HiSeq 2500   SRP455680     Fish_embryo_mRNA_germ_ring_PAL_seq_v4_rep2_raw_read1.fastq.gz Fish_embryo_mRNA_germ_ring_PAL_seq_v4_rep2_raw_read2.fastq.gz fastq fastq 3473033898.0 11312814.0 GSM7716871 r2 0:52 1:255 A:853178471;C:899976159;G:962100712;T:750811461;N:6967095 52 255     853178471 899976159 962100712 750811461 6967095 SRX21396042 SRS18636200 SRA1694849 Whitehead Institute Whitehead Institute 2 0.00049 0.0 0.00012 0.0 0.99941 1.0 0.64864   52 255 T T mates < 9% mapping rate illumina hiseq_era unknown poly_a unknown bulk bulk bulk   United States 2023-08-17 Multi-stage Embryo Embryo Imprecise All anatomical structures
25184 25184 SRR25670731 SRX21396041 SRS18636199 SRP455680 PRJNA1006406 Control of polyA tail length and translation in vertebrate oocytes and early embryos GSE241107 Other During oocyte maturation and early embryonic development polyA tail lengths strongly influence mRNA translation. However how tail lengths are controlled at different developmental stages has been unclear. Here we performed tail length and translational profiling of mRNA reporter libraries each with > 10 million three prime UTR sequence variants in frog oocytes and embryos and fish embryos. These analyses revealed that the UUUUA motif specifies cytoplasmic polyadenylation and identified diverse context features that modulate the activity of this 5 mer. Additional sequence motifs drive stage specific deadenylation in embryos and UUUUA and C rich motifs drive tail length independent translational repression in oocytes. A neural network model accurately predicts tail length change during oocyte maturation in frogs mice and humans. Analyses of human sequence variants showed that those predicted to disrupt tail length control have been under negative selection implying that our insights into control of polyA tail length and translation have implications for human health and fertility. Overall design: Sythetic mRNA reporter libraries with random three prime UTR sequences under four different sequence contexts were injected into frog oocytes and embryos and fish embryos. PolyA tail lengths were measured to at different developmental stages to examine sequence motifs that caused tail length changes. At the same time polyA tail lengths of endogenous mRNAs from frog oocytes and embryos fish embryos and mouse oocytes were measured to investiage how their tail lengths were controlled. Please note that sample titles have been updated on Jan 6 2024.       Fish embryo mRNA zfs:0000015 PAL seq v4 GSM7716870   source name:embryo|tissue:embryo|treatment:N1|geo loc name:missing|collection date:missing Fish embryo mRNA zfs:0000015 PAL seq v4 For gene specific tail seq of mRNA reporters data were processed with a custom script available at https://github.com/coffeebond/MPRA tail seq. For PAL seq v3 or v4 reads were trimmed with cutadapt v3.7 with the parameters “ m 15 quality base=64 q 20 20 match read wildcards e 0.05 a NNNNATCTCGTATGCCGTCTTCTGCTTG O 7”. The trimmed reads were mapped using STAR v2.7.1a to the reference database containing the genomic sequences of the organism from which the mRNAs were obtained the genomic sequences of humans or fish depending on which spike in RNAs were used and the sequences of the polyA standards generated previously with the parameters “ runThreadN 16 runMode alignReads outFilterMultimapNmax 1 outReadsUnmapped Fastx outFilterType BySJout outSAMattributes All outSAMtype BAM Unsorted SortedByCoordinate”. Uniquely mapped read were furhter processed with a custom script available at https://github.com/coffeebond/PAL seq. Assembly: Homo sapiens: GRCh38.p7 primary assembly. Mus muculus: GRCh38.p4 primary assembly. Xenopus laevis: v10.1 assembly. Danio rerio: GRCz11 assembly. Supplementary files format and content: For gene specific tail seq of mRNA reporters tab delimited text files with columns indicating 1 sequence of the variable region; 2 median tail length; 3 number of reads; 4–7 tail lengths at quantile 10 25 75 and 90. Supplementary files format and content: For PAL seq v3 or v4 tab delimited files with columns indicating 1 gene ID or polyA site ID; 2 read cluster ID; 3 tail length Library strategy: PAL seq v4 embryo   Frog oocytes and embryos were lysed in ice cold buffer RL 20 mM HEPES pH 7.5 100 mM KCl 5 mM MgCl2 1% [v/v] Triton X 100 100 µg/ml cycloheximide cOmplete protease inhibitor cocktail [1 tablet per 10 ml buffer] and 200 units/ml SUPERase•In in a volume of 10 µl per oocyte/embryo by vigorous shaking and pipetting. Lysates were cleared by centrifugation at 5000 g at 4°C for 10 min. The supernatant was transfered to a new tube and mixed with the Tri reagent for RNA isolation. Fish embryos were de chorionated by incubation with 2 mg/ml pronase in E3 medium for 4 min. post removing all E3 medium Tri Reagent was added to the embryos for RNA isolation. Mouse GV oocytes were collected in 37°C MEM in the presence of milrinone to prevent maturation. Cumulus cells were removed from cumulus oocyte complexes by repeated aspiration through a glass pipette. GV oocytes were then collected in TRI reagent for RNA isolation. Mouse MII oocytes were harvested from the oviducts of hCG induced mice 16 hr denuded with 3 mg/ml hyaluronidase for 2 min washed and then collected in Tri reagent for RNA isolation. For gene specific tail seq of reporter libraries total RNA with reporter mRNA libraries was ligated to a pre adenylated 3ʹ adapter directly in most cases but for the N60 library injected into fish embryos and frog oocytes the N37 PAS N17 library injected into fish embryos and frog oocytes and the CPEmos N60 and N60LC PASmos libraries injected into frog oocytes reporter library mRNAs were enriched by anti sense oligo capture with biotinylated oligos. RNA isolated from the oocyte or embryo lysate was mixed with 8 pmol KXSH009 8 pmol KXSH010 and 2x SSC 0.3 M NaCl 30 mM sodium citrate pH 7.0 in a total of 50 µl. The RNA and oligos were annealed by incubation at 70°C for 5 min and then slowly cooling to 23°C at 0.1°C/sec. The annealed mixture was combined with 40 µl MyOne Streptavidin C1 beads Thermo Fisher 65002 and incubated for 20 min at 23°C on a thermal mixer shaking with 15 sec on and 1 min 45 sec off. The superna…   tissue:embryo|treatment:N1 GSM7716870 GSM7716870: Fish embryo mRNA zfs:0000015 PAL seq v4; Danio rerio; OTHER GSM7716870 r1 GSM7716870 1 Frog oocytes and embryos were lysed in ice cold buffer RL 20 mM HEPES pH 7.5 100 mM KCl 5 mM MgCl2 1% [v/v] Triton X 100 100 µg/ml cycloheximide cOmplete protease inhibitor cocktail [1 tablet per 10 ml buffer] and 200 units/ml SUPERase•In in a volume of 10 µl per oocyte/embryo by vigorous shaking and pipetting. Lysates were cleared by centrifugation at 5000 g at 4°C for 10 min. The supernatant was transfered to a new tube and mixed with the Tri reagent for RNA isolation. Fish embryos were de chorionated by incubation with 2 mg/ml pronase in E3 medium for 4 min. post removing all E3 medium Tri Reagent was added to the embryos for RNA isolation. Mouse GV oocytes were collected in 37°C MEM in the presence of milrinone to prevent maturation. Cumulus cells were removed from cumulus oocyte complexes by repeated aspiration through a glass pipette. GV oocytes were then collected in TRI reagent for RNA isolation. Mouse MII oocytes were harvested from the oviducts of hCG induced mice 16 hr denuded with 3 mg/ml hyaluronidase for 2 min washed and then collected in Tri reagent for RNA isolation. For gene specific tail seq of reporter libraries total RNA with reporter mRNA libraries was ligated to a pre adenylated 3ʹ adapter directly in most cases but for the N60 library injected into fish embryos and frog oocytes the N37 PAS N17 library injected into fish embryos and frog oocytes and the CPEmos N60 and N60LC PASmos libraries injected into frog oocytes reporter library mRNAs were enriched by anti sense oligo capture with biotinylated oligos. RNA isolated from the oocyte or embryo lysate was mixed with 8 pmol KXSH009 8 pmol KXSH010 and 2x SSC 0.3 M NaCl 30 mM sodium citrate pH 7.0 in a total of 50 µl. The RNA and oligos were annealed by incubation at 70°C for 5 min and then slowly cooling to 23°C at 0.1°C/sec. The annealed mixture was combined with 40 µl MyOne Streptavidin C1 beads Thermo Fisher 65002 and incubated for 20 min at 23°C on a thermal mixer shaking with 15 sec on and 1 min 45 sec off. The superna…   OTHER TRANSCRIPTOMIC other PAIRED ILLUMINA Illumina HiSeq 2500   SRP455680     Fish_embryo_mRNA_30percent_epiboly_PAL_seq_v4_rep1_raw_read1.fastq.gz Fish_embryo_mRNA_30percent_epiboly_PAL_seq_v4_rep1_raw_read2.fastq.gz fastq fastq 2695203962.0 8779166.0 GSM7716870 r1 0:52 1:255 A:684535386;C:669852151;G:719008886;T:614209811;N:7597728 52 255     684535386 669852151 719008886 614209811 7597728 SRX21396041 SRS18636199 SRA1694849 Whitehead Institute Whitehead Institute 2 0.00117 0.35837 0.00017 0.00682 0.99859 0.99963 0.69473 1.0 52 255 T B mate1 technical by mapping diff illumina hiseq_era unknown poly_a unknown bulk bulk bulk   United States 2023-08-17 Multi-stage Embryo Embryo Imprecise All anatomical structures
25185 25185 SRR25670732 SRX21396041 SRS18636199 SRP455680 PRJNA1006406 Control of polyA tail length and translation in vertebrate oocytes and early embryos GSE241107 Other During oocyte maturation and early embryonic development polyA tail lengths strongly influence mRNA translation. However how tail lengths are controlled at different developmental stages has been unclear. Here we performed tail length and translational profiling of mRNA reporter libraries each with > 10 million three prime UTR sequence variants in frog oocytes and embryos and fish embryos. These analyses revealed that the UUUUA motif specifies cytoplasmic polyadenylation and identified diverse context features that modulate the activity of this 5 mer. Additional sequence motifs drive stage specific deadenylation in embryos and UUUUA and C rich motifs drive tail length independent translational repression in oocytes. A neural network model accurately predicts tail length change during oocyte maturation in frogs mice and humans. Analyses of human sequence variants showed that those predicted to disrupt tail length control have been under negative selection implying that our insights into control of polyA tail length and translation have implications for human health and fertility. Overall design: Sythetic mRNA reporter libraries with random three prime UTR sequences under four different sequence contexts were injected into frog oocytes and embryos and fish embryos. PolyA tail lengths were measured to at different developmental stages to examine sequence motifs that caused tail length changes. At the same time polyA tail lengths of endogenous mRNAs from frog oocytes and embryos fish embryos and mouse oocytes were measured to investiage how their tail lengths were controlled. Please note that sample titles have been updated on Jan 6 2024.       Fish embryo mRNA zfs:0000015 PAL seq v4 GSM7716870   source name:embryo|tissue:embryo|treatment:N1|geo loc name:missing|collection date:missing Fish embryo mRNA zfs:0000015 PAL seq v4 For gene specific tail seq of mRNA reporters data were processed with a custom script available at https://github.com/coffeebond/MPRA tail seq. For PAL seq v3 or v4 reads were trimmed with cutadapt v3.7 with the parameters “ m 15 quality base=64 q 20 20 match read wildcards e 0.05 a NNNNATCTCGTATGCCGTCTTCTGCTTG O 7”. The trimmed reads were mapped using STAR v2.7.1a to the reference database containing the genomic sequences of the organism from which the mRNAs were obtained the genomic sequences of humans or fish depending on which spike in RNAs were used and the sequences of the polyA standards generated previously with the parameters “ runThreadN 16 runMode alignReads outFilterMultimapNmax 1 outReadsUnmapped Fastx outFilterType BySJout outSAMattributes All outSAMtype BAM Unsorted SortedByCoordinate”. Uniquely mapped read were furhter processed with a custom script available at https://github.com/coffeebond/PAL seq. Assembly: Homo sapiens: GRCh38.p7 primary assembly. Mus muculus: GRCh38.p4 primary assembly. Xenopus laevis: v10.1 assembly. Danio rerio: GRCz11 assembly. Supplementary files format and content: For gene specific tail seq of mRNA reporters tab delimited text files with columns indicating 1 sequence of the variable region; 2 median tail length; 3 number of reads; 4–7 tail lengths at quantile 10 25 75 and 90. Supplementary files format and content: For PAL seq v3 or v4 tab delimited files with columns indicating 1 gene ID or polyA site ID; 2 read cluster ID; 3 tail length Library strategy: PAL seq v4 embryo   Frog oocytes and embryos were lysed in ice cold buffer RL 20 mM HEPES pH 7.5 100 mM KCl 5 mM MgCl2 1% [v/v] Triton X 100 100 µg/ml cycloheximide cOmplete protease inhibitor cocktail [1 tablet per 10 ml buffer] and 200 units/ml SUPERase•In in a volume of 10 µl per oocyte/embryo by vigorous shaking and pipetting. Lysates were cleared by centrifugation at 5000 g at 4°C for 10 min. The supernatant was transfered to a new tube and mixed with the Tri reagent for RNA isolation. Fish embryos were de chorionated by incubation with 2 mg/ml pronase in E3 medium for 4 min. post removing all E3 medium Tri Reagent was added to the embryos for RNA isolation. Mouse GV oocytes were collected in 37°C MEM in the presence of milrinone to prevent maturation. Cumulus cells were removed from cumulus oocyte complexes by repeated aspiration through a glass pipette. GV oocytes were then collected in TRI reagent for RNA isolation. Mouse MII oocytes were harvested from the oviducts of hCG induced mice 16 hr denuded with 3 mg/ml hyaluronidase for 2 min washed and then collected in Tri reagent for RNA isolation. For gene specific tail seq of reporter libraries total RNA with reporter mRNA libraries was ligated to a pre adenylated 3ʹ adapter directly in most cases but for the N60 library injected into fish embryos and frog oocytes the N37 PAS N17 library injected into fish embryos and frog oocytes and the CPEmos N60 and N60LC PASmos libraries injected into frog oocytes reporter library mRNAs were enriched by anti sense oligo capture with biotinylated oligos. RNA isolated from the oocyte or embryo lysate was mixed with 8 pmol KXSH009 8 pmol KXSH010 and 2x SSC 0.3 M NaCl 30 mM sodium citrate pH 7.0 in a total of 50 µl. The RNA and oligos were annealed by incubation at 70°C for 5 min and then slowly cooling to 23°C at 0.1°C/sec. The annealed mixture was combined with 40 µl MyOne Streptavidin C1 beads Thermo Fisher 65002 and incubated for 20 min at 23°C on a thermal mixer shaking with 15 sec on and 1 min 45 sec off. The superna…   tissue:embryo|treatment:N1 GSM7716870 GSM7716870: Fish embryo mRNA zfs:0000015 PAL seq v4; Danio rerio; OTHER GSM7716870 r1 GSM7716870 1 Frog oocytes and embryos were lysed in ice cold buffer RL 20 mM HEPES pH 7.5 100 mM KCl 5 mM MgCl2 1% [v/v] Triton X 100 100 µg/ml cycloheximide cOmplete protease inhibitor cocktail [1 tablet per 10 ml buffer] and 200 units/ml SUPERase•In in a volume of 10 µl per oocyte/embryo by vigorous shaking and pipetting. Lysates were cleared by centrifugation at 5000 g at 4°C for 10 min. The supernatant was transfered to a new tube and mixed with the Tri reagent for RNA isolation. Fish embryos were de chorionated by incubation with 2 mg/ml pronase in E3 medium for 4 min. post removing all E3 medium Tri Reagent was added to the embryos for RNA isolation. Mouse GV oocytes were collected in 37°C MEM in the presence of milrinone to prevent maturation. Cumulus cells were removed from cumulus oocyte complexes by repeated aspiration through a glass pipette. GV oocytes were then collected in TRI reagent for RNA isolation. Mouse MII oocytes were harvested from the oviducts of hCG induced mice 16 hr denuded with 3 mg/ml hyaluronidase for 2 min washed and then collected in Tri reagent for RNA isolation. For gene specific tail seq of reporter libraries total RNA with reporter mRNA libraries was ligated to a pre adenylated 3ʹ adapter directly in most cases but for the N60 library injected into fish embryos and frog oocytes the N37 PAS N17 library injected into fish embryos and frog oocytes and the CPEmos N60 and N60LC PASmos libraries injected into frog oocytes reporter library mRNAs were enriched by anti sense oligo capture with biotinylated oligos. RNA isolated from the oocyte or embryo lysate was mixed with 8 pmol KXSH009 8 pmol KXSH010 and 2x SSC 0.3 M NaCl 30 mM sodium citrate pH 7.0 in a total of 50 µl. The RNA and oligos were annealed by incubation at 70°C for 5 min and then slowly cooling to 23°C at 0.1°C/sec. The annealed mixture was combined with 40 µl MyOne Streptavidin C1 beads Thermo Fisher 65002 and incubated for 20 min at 23°C on a thermal mixer shaking with 15 sec on and 1 min 45 sec off. The superna…   OTHER TRANSCRIPTOMIC other PAIRED ILLUMINA Illumina HiSeq 2500   SRP455680     Fish_embryo_mRNA_30percent_epiboly_PAL_seq_v4_rep2_raw_read2.fastq.gz Fish_embryo_mRNA_30percent_epiboly_PAL_seq_v4_rep2_raw_read1.fastq.gz fastq fastq 3476338446.0 11323578.0 GSM7716870 r2 0:52 1:255 A:841832234;C:898325199;G:968774318;T:760398070;N:7008625 52 255     841832234 898325199 968774318 760398070 7008625 SRX21396041 SRS18636199 SRA1694849 Whitehead Institute Whitehead Institute 2 0.00245 0.0 0.00047 0.0 0.99803 1.0 0.58536   52 255 T T mates < 9% mapping rate illumina hiseq_era unknown poly_a unknown bulk bulk bulk   United States 2023-08-17 Multi-stage Embryo Embryo Imprecise All anatomical structures
25186 25186 SRR25670733 SRX21396040 SRS18636198 SRP455680 PRJNA1006406 Control of polyA tail length and translation in vertebrate oocytes and early embryos GSE241107 Other During oocyte maturation and early embryonic development polyA tail lengths strongly influence mRNA translation. However how tail lengths are controlled at different developmental stages has been unclear. Here we performed tail length and translational profiling of mRNA reporter libraries each with > 10 million three prime UTR sequence variants in frog oocytes and embryos and fish embryos. These analyses revealed that the UUUUA motif specifies cytoplasmic polyadenylation and identified diverse context features that modulate the activity of this 5 mer. Additional sequence motifs drive stage specific deadenylation in embryos and UUUUA and C rich motifs drive tail length independent translational repression in oocytes. A neural network model accurately predicts tail length change during oocyte maturation in frogs mice and humans. Analyses of human sequence variants showed that those predicted to disrupt tail length control have been under negative selection implying that our insights into control of polyA tail length and translation have implications for human health and fertility. Overall design: Sythetic mRNA reporter libraries with random three prime UTR sequences under four different sequence contexts were injected into frog oocytes and embryos and fish embryos. PolyA tail lengths were measured to at different developmental stages to examine sequence motifs that caused tail length changes. At the same time polyA tail lengths of endogenous mRNAs from frog oocytes and embryos fish embryos and mouse oocytes were measured to investiage how their tail lengths were controlled. Please note that sample titles have been updated on Jan 6 2024.       Fish embryo mRNA sphere PAL seq v4 GSM7716869   source name:embryo|tissue:embryo|treatment:N1|geo loc name:missing|collection date:missing Fish embryo mRNA sphere PAL seq v4 For gene specific tail seq of mRNA reporters data were processed with a custom script available at https://github.com/coffeebond/MPRA tail seq. For PAL seq v3 or v4 reads were trimmed with cutadapt v3.7 with the parameters “ m 15 quality base=64 q 20 20 match read wildcards e 0.05 a NNNNATCTCGTATGCCGTCTTCTGCTTG O 7”. The trimmed reads were mapped using STAR v2.7.1a to the reference database containing the genomic sequences of the organism from which the mRNAs were obtained the genomic sequences of humans or fish depending on which spike in RNAs were used and the sequences of the polyA standards generated previously with the parameters “ runThreadN 16 runMode alignReads outFilterMultimapNmax 1 outReadsUnmapped Fastx outFilterType BySJout outSAMattributes All outSAMtype BAM Unsorted SortedByCoordinate”. Uniquely mapped read were furhter processed with a custom script available at https://github.com/coffeebond/PAL seq. Assembly: Homo sapiens: GRCh38.p7 primary assembly. Mus muculus: GRCh38.p4 primary assembly. Xenopus laevis: v10.1 assembly. Danio rerio: GRCz11 assembly. Supplementary files format and content: For gene specific tail seq of mRNA reporters tab delimited text files with columns indicating 1 sequence of the variable region; 2 median tail length; 3 number of reads; 4–7 tail lengths at quantile 10 25 75 and 90. Supplementary files format and content: For PAL seq v3 or v4 tab delimited files with columns indicating 1 gene ID or polyA site ID; 2 read cluster ID; 3 tail length Library strategy: PAL seq v4 embryo   Frog oocytes and embryos were lysed in ice cold buffer RL 20 mM HEPES pH 7.5 100 mM KCl 5 mM MgCl2 1% [v/v] Triton X 100 100 µg/ml cycloheximide cOmplete protease inhibitor cocktail [1 tablet per 10 ml buffer] and 200 units/ml SUPERase•In in a volume of 10 µl per oocyte/embryo by vigorous shaking and pipetting. Lysates were cleared by centrifugation at 5000 g at 4°C for 10 min. The supernatant was transfered to a new tube and mixed with the Tri reagent for RNA isolation. Fish embryos were de chorionated by incubation with 2 mg/ml pronase in E3 medium for 4 min. post removing all E3 medium Tri Reagent was added to the embryos for RNA isolation. Mouse GV oocytes were collected in 37°C MEM in the presence of milrinone to prevent maturation. Cumulus cells were removed from cumulus oocyte complexes by repeated aspiration through a glass pipette. GV oocytes were then collected in TRI reagent for RNA isolation. Mouse MII oocytes were harvested from the oviducts of hCG induced mice 16 hr denuded with 3 mg/ml hyaluronidase for 2 min washed and then collected in Tri reagent for RNA isolation. For gene specific tail seq of reporter libraries total RNA with reporter mRNA libraries was ligated to a pre adenylated 3ʹ adapter directly in most cases but for the N60 library injected into fish embryos and frog oocytes the N37 PAS N17 library injected into fish embryos and frog oocytes and the CPEmos N60 and N60LC PASmos libraries injected into frog oocytes reporter library mRNAs were enriched by anti sense oligo capture with biotinylated oligos. RNA isolated from the oocyte or embryo lysate was mixed with 8 pmol KXSH009 8 pmol KXSH010 and 2x SSC 0.3 M NaCl 30 mM sodium citrate pH 7.0 in a total of 50 µl. The RNA and oligos were annealed by incubation at 70°C for 5 min and then slowly cooling to 23°C at 0.1°C/sec. The annealed mixture was combined with 40 µl MyOne Streptavidin C1 beads Thermo Fisher 65002 and incubated for 20 min at 23°C on a thermal mixer shaking with 15 sec on and 1 min 45 sec off. The superna…   tissue:embryo|treatment:N1 GSM7716869 GSM7716869: Fish embryo mRNA sphere PAL seq v4; Danio rerio; OTHER GSM7716869 r1 GSM7716869 1 Frog oocytes and embryos were lysed in ice cold buffer RL 20 mM HEPES pH 7.5 100 mM KCl 5 mM MgCl2 1% [v/v] Triton X 100 100 µg/ml cycloheximide cOmplete protease inhibitor cocktail [1 tablet per 10 ml buffer] and 200 units/ml SUPERase•In in a volume of 10 µl per oocyte/embryo by vigorous shaking and pipetting. Lysates were cleared by centrifugation at 5000 g at 4°C for 10 min. The supernatant was transfered to a new tube and mixed with the Tri reagent for RNA isolation. Fish embryos were de chorionated by incubation with 2 mg/ml pronase in E3 medium for 4 min. post removing all E3 medium Tri Reagent was added to the embryos for RNA isolation. Mouse GV oocytes were collected in 37°C MEM in the presence of milrinone to prevent maturation. Cumulus cells were removed from cumulus oocyte complexes by repeated aspiration through a glass pipette. GV oocytes were then collected in TRI reagent for RNA isolation. Mouse MII oocytes were harvested from the oviducts of hCG induced mice 16 hr denuded with 3 mg/ml hyaluronidase for 2 min washed and then collected in Tri reagent for RNA isolation. For gene specific tail seq of reporter libraries total RNA with reporter mRNA libraries was ligated to a pre adenylated 3ʹ adapter directly in most cases but for the N60 library injected into fish embryos and frog oocytes the N37 PAS N17 library injected into fish embryos and frog oocytes and the CPEmos N60 and N60LC PASmos libraries injected into frog oocytes reporter library mRNAs were enriched by anti sense oligo capture with biotinylated oligos. RNA isolated from the oocyte or embryo lysate was mixed with 8 pmol KXSH009 8 pmol KXSH010 and 2x SSC 0.3 M NaCl 30 mM sodium citrate pH 7.0 in a total of 50 µl. The RNA and oligos were annealed by incubation at 70°C for 5 min and then slowly cooling to 23°C at 0.1°C/sec. The annealed mixture was combined with 40 µl MyOne Streptavidin C1 beads Thermo Fisher 65002 and incubated for 20 min at 23°C on a thermal mixer shaking with 15 sec on and 1 min 45 sec off. The superna…   OTHER TRANSCRIPTOMIC other PAIRED ILLUMINA Illumina HiSeq 2500   SRP455680     Fish_embryo_mRNA_sphere_PAL_seq_v4_rep1_raw_read1.fastq.gz Fish_embryo_mRNA_sphere_PAL_seq_v4_rep1_raw_read2.fastq.gz fastq fastq 3211687254.0 10461522.0 GSM7716869 r1 0:52 1:255 A:813560400;C:775326762;G:854563477;T:759161458;N:9075157 52 255     813560400 775326762 854563477 759161458 9075157 SRX21396040 SRS18636198 SRA1694849 Whitehead Institute Whitehead Institute 2 0.00088 0.43191 0.0004 0.01556 0.99916 0.99961 0.55769 1.0 52 255 T B mate1 technical by mapping diff illumina hiseq_era unknown poly_a unknown bulk bulk bulk   United States 2023-08-17 Multi-stage Embryo Embryo Imprecise All anatomical structures
25187 25187 SRR25670734 SRX21396040 SRS18636198 SRP455680 PRJNA1006406 Control of polyA tail length and translation in vertebrate oocytes and early embryos GSE241107 Other During oocyte maturation and early embryonic development polyA tail lengths strongly influence mRNA translation. However how tail lengths are controlled at different developmental stages has been unclear. Here we performed tail length and translational profiling of mRNA reporter libraries each with > 10 million three prime UTR sequence variants in frog oocytes and embryos and fish embryos. These analyses revealed that the UUUUA motif specifies cytoplasmic polyadenylation and identified diverse context features that modulate the activity of this 5 mer. Additional sequence motifs drive stage specific deadenylation in embryos and UUUUA and C rich motifs drive tail length independent translational repression in oocytes. A neural network model accurately predicts tail length change during oocyte maturation in frogs mice and humans. Analyses of human sequence variants showed that those predicted to disrupt tail length control have been under negative selection implying that our insights into control of polyA tail length and translation have implications for human health and fertility. Overall design: Sythetic mRNA reporter libraries with random three prime UTR sequences under four different sequence contexts were injected into frog oocytes and embryos and fish embryos. PolyA tail lengths were measured to at different developmental stages to examine sequence motifs that caused tail length changes. At the same time polyA tail lengths of endogenous mRNAs from frog oocytes and embryos fish embryos and mouse oocytes were measured to investiage how their tail lengths were controlled. Please note that sample titles have been updated on Jan 6 2024.       Fish embryo mRNA sphere PAL seq v4 GSM7716869   source name:embryo|tissue:embryo|treatment:N1|geo loc name:missing|collection date:missing Fish embryo mRNA sphere PAL seq v4 For gene specific tail seq of mRNA reporters data were processed with a custom script available at https://github.com/coffeebond/MPRA tail seq. For PAL seq v3 or v4 reads were trimmed with cutadapt v3.7 with the parameters “ m 15 quality base=64 q 20 20 match read wildcards e 0.05 a NNNNATCTCGTATGCCGTCTTCTGCTTG O 7”. The trimmed reads were mapped using STAR v2.7.1a to the reference database containing the genomic sequences of the organism from which the mRNAs were obtained the genomic sequences of humans or fish depending on which spike in RNAs were used and the sequences of the polyA standards generated previously with the parameters “ runThreadN 16 runMode alignReads outFilterMultimapNmax 1 outReadsUnmapped Fastx outFilterType BySJout outSAMattributes All outSAMtype BAM Unsorted SortedByCoordinate”. Uniquely mapped read were furhter processed with a custom script available at https://github.com/coffeebond/PAL seq. Assembly: Homo sapiens: GRCh38.p7 primary assembly. Mus muculus: GRCh38.p4 primary assembly. Xenopus laevis: v10.1 assembly. Danio rerio: GRCz11 assembly. Supplementary files format and content: For gene specific tail seq of mRNA reporters tab delimited text files with columns indicating 1 sequence of the variable region; 2 median tail length; 3 number of reads; 4–7 tail lengths at quantile 10 25 75 and 90. Supplementary files format and content: For PAL seq v3 or v4 tab delimited files with columns indicating 1 gene ID or polyA site ID; 2 read cluster ID; 3 tail length Library strategy: PAL seq v4 embryo   Frog oocytes and embryos were lysed in ice cold buffer RL 20 mM HEPES pH 7.5 100 mM KCl 5 mM MgCl2 1% [v/v] Triton X 100 100 µg/ml cycloheximide cOmplete protease inhibitor cocktail [1 tablet per 10 ml buffer] and 200 units/ml SUPERase•In in a volume of 10 µl per oocyte/embryo by vigorous shaking and pipetting. Lysates were cleared by centrifugation at 5000 g at 4°C for 10 min. The supernatant was transfered to a new tube and mixed with the Tri reagent for RNA isolation. Fish embryos were de chorionated by incubation with 2 mg/ml pronase in E3 medium for 4 min. post removing all E3 medium Tri Reagent was added to the embryos for RNA isolation. Mouse GV oocytes were collected in 37°C MEM in the presence of milrinone to prevent maturation. Cumulus cells were removed from cumulus oocyte complexes by repeated aspiration through a glass pipette. GV oocytes were then collected in TRI reagent for RNA isolation. Mouse MII oocytes were harvested from the oviducts of hCG induced mice 16 hr denuded with 3 mg/ml hyaluronidase for 2 min washed and then collected in Tri reagent for RNA isolation. For gene specific tail seq of reporter libraries total RNA with reporter mRNA libraries was ligated to a pre adenylated 3ʹ adapter directly in most cases but for the N60 library injected into fish embryos and frog oocytes the N37 PAS N17 library injected into fish embryos and frog oocytes and the CPEmos N60 and N60LC PASmos libraries injected into frog oocytes reporter library mRNAs were enriched by anti sense oligo capture with biotinylated oligos. RNA isolated from the oocyte or embryo lysate was mixed with 8 pmol KXSH009 8 pmol KXSH010 and 2x SSC 0.3 M NaCl 30 mM sodium citrate pH 7.0 in a total of 50 µl. The RNA and oligos were annealed by incubation at 70°C for 5 min and then slowly cooling to 23°C at 0.1°C/sec. The annealed mixture was combined with 40 µl MyOne Streptavidin C1 beads Thermo Fisher 65002 and incubated for 20 min at 23°C on a thermal mixer shaking with 15 sec on and 1 min 45 sec off. The superna…   tissue:embryo|treatment:N1 GSM7716869 GSM7716869: Fish embryo mRNA sphere PAL seq v4; Danio rerio; OTHER GSM7716869 r1 GSM7716869 1 Frog oocytes and embryos were lysed in ice cold buffer RL 20 mM HEPES pH 7.5 100 mM KCl 5 mM MgCl2 1% [v/v] Triton X 100 100 µg/ml cycloheximide cOmplete protease inhibitor cocktail [1 tablet per 10 ml buffer] and 200 units/ml SUPERase•In in a volume of 10 µl per oocyte/embryo by vigorous shaking and pipetting. Lysates were cleared by centrifugation at 5000 g at 4°C for 10 min. The supernatant was transfered to a new tube and mixed with the Tri reagent for RNA isolation. Fish embryos were de chorionated by incubation with 2 mg/ml pronase in E3 medium for 4 min. post removing all E3 medium Tri Reagent was added to the embryos for RNA isolation. Mouse GV oocytes were collected in 37°C MEM in the presence of milrinone to prevent maturation. Cumulus cells were removed from cumulus oocyte complexes by repeated aspiration through a glass pipette. GV oocytes were then collected in TRI reagent for RNA isolation. Mouse MII oocytes were harvested from the oviducts of hCG induced mice 16 hr denuded with 3 mg/ml hyaluronidase for 2 min washed and then collected in Tri reagent for RNA isolation. For gene specific tail seq of reporter libraries total RNA with reporter mRNA libraries was ligated to a pre adenylated 3ʹ adapter directly in most cases but for the N60 library injected into fish embryos and frog oocytes the N37 PAS N17 library injected into fish embryos and frog oocytes and the CPEmos N60 and N60LC PASmos libraries injected into frog oocytes reporter library mRNAs were enriched by anti sense oligo capture with biotinylated oligos. RNA isolated from the oocyte or embryo lysate was mixed with 8 pmol KXSH009 8 pmol KXSH010 and 2x SSC 0.3 M NaCl 30 mM sodium citrate pH 7.0 in a total of 50 µl. The RNA and oligos were annealed by incubation at 70°C for 5 min and then slowly cooling to 23°C at 0.1°C/sec. The annealed mixture was combined with 40 µl MyOne Streptavidin C1 beads Thermo Fisher 65002 and incubated for 20 min at 23°C on a thermal mixer shaking with 15 sec on and 1 min 45 sec off. The superna…   OTHER TRANSCRIPTOMIC other PAIRED ILLUMINA Illumina HiSeq 2500   SRP455680     Fish_embryo_mRNA_sphere_PAL_seq_v4_rep2_raw_read1.fastq.gz Fish_embryo_mRNA_sphere_PAL_seq_v4_rep2_raw_read2.fastq.gz fastq fastq 3179916131.0 10358033.0 GSM7716869 r2 0:52 1:255 A:771755667;C:804256300;G:883166175;T:714242073;N:6495916 52 255     771755667 804256300 883166175 714242073 6495916 SRX21396040 SRS18636198 SRA1694849 Whitehead Institute Whitehead Institute 2 0.00186 0.0 0.00088 0.0 0.99862 1.0 0.64705   52 255 T T mates < 9% mapping rate illumina hiseq_era unknown poly_a unknown bulk bulk bulk   United States 2023-08-17 Multi-stage Embryo Embryo Imprecise All anatomical structures
25188 25188 SRR25670735 SRX21396039 SRS18636197 SRP455680 PRJNA1006406 Control of polyA tail length and translation in vertebrate oocytes and early embryos GSE241107 Other During oocyte maturation and early embryonic development polyA tail lengths strongly influence mRNA translation. However how tail lengths are controlled at different developmental stages has been unclear. Here we performed tail length and translational profiling of mRNA reporter libraries each with > 10 million three prime UTR sequence variants in frog oocytes and embryos and fish embryos. These analyses revealed that the UUUUA motif specifies cytoplasmic polyadenylation and identified diverse context features that modulate the activity of this 5 mer. Additional sequence motifs drive stage specific deadenylation in embryos and UUUUA and C rich motifs drive tail length independent translational repression in oocytes. A neural network model accurately predicts tail length change during oocyte maturation in frogs mice and humans. Analyses of human sequence variants showed that those predicted to disrupt tail length control have been under negative selection implying that our insights into control of polyA tail length and translation have implications for human health and fertility. Overall design: Sythetic mRNA reporter libraries with random three prime UTR sequences under four different sequence contexts were injected into frog oocytes and embryos and fish embryos. PolyA tail lengths were measured to at different developmental stages to examine sequence motifs that caused tail length changes. At the same time polyA tail lengths of endogenous mRNAs from frog oocytes and embryos fish embryos and mouse oocytes were measured to investiage how their tail lengths were controlled. Please note that sample titles have been updated on Jan 6 2024.       Fish embryo mRNA 1024cell PAL seq v4 GSM7716868   source name:embryo|tissue:embryo|treatment:N1|geo loc name:missing|collection date:missing Fish embryo mRNA 1024cell PAL seq v4 For gene specific tail seq of mRNA reporters data were processed with a custom script available at https://github.com/coffeebond/MPRA tail seq. For PAL seq v3 or v4 reads were trimmed with cutadapt v3.7 with the parameters “ m 15 quality base=64 q 20 20 match read wildcards e 0.05 a NNNNATCTCGTATGCCGTCTTCTGCTTG O 7”. The trimmed reads were mapped using STAR v2.7.1a to the reference database containing the genomic sequences of the organism from which the mRNAs were obtained the genomic sequences of humans or fish depending on which spike in RNAs were used and the sequences of the polyA standards generated previously with the parameters “ runThreadN 16 runMode alignReads outFilterMultimapNmax 1 outReadsUnmapped Fastx outFilterType BySJout outSAMattributes All outSAMtype BAM Unsorted SortedByCoordinate”. Uniquely mapped read were furhter processed with a custom script available at https://github.com/coffeebond/PAL seq. Assembly: Homo sapiens: GRCh38.p7 primary assembly. Mus muculus: GRCh38.p4 primary assembly. Xenopus laevis: v10.1 assembly. Danio rerio: GRCz11 assembly. Supplementary files format and content: For gene specific tail seq of mRNA reporters tab delimited text files with columns indicating 1 sequence of the variable region; 2 median tail length; 3 number of reads; 4–7 tail lengths at quantile 10 25 75 and 90. Supplementary files format and content: For PAL seq v3 or v4 tab delimited files with columns indicating 1 gene ID or polyA site ID; 2 read cluster ID; 3 tail length Library strategy: PAL seq v4 embryo   Frog oocytes and embryos were lysed in ice cold buffer RL 20 mM HEPES pH 7.5 100 mM KCl 5 mM MgCl2 1% [v/v] Triton X 100 100 µg/ml cycloheximide cOmplete protease inhibitor cocktail [1 tablet per 10 ml buffer] and 200 units/ml SUPERase•In in a volume of 10 µl per oocyte/embryo by vigorous shaking and pipetting. Lysates were cleared by centrifugation at 5000 g at 4°C for 10 min. The supernatant was transfered to a new tube and mixed with the Tri reagent for RNA isolation. Fish embryos were de chorionated by incubation with 2 mg/ml pronase in E3 medium for 4 min. post removing all E3 medium Tri Reagent was added to the embryos for RNA isolation. Mouse GV oocytes were collected in 37°C MEM in the presence of milrinone to prevent maturation. Cumulus cells were removed from cumulus oocyte complexes by repeated aspiration through a glass pipette. GV oocytes were then collected in TRI reagent for RNA isolation. Mouse MII oocytes were harvested from the oviducts of hCG induced mice 16 hr denuded with 3 mg/ml hyaluronidase for 2 min washed and then collected in Tri reagent for RNA isolation. For gene specific tail seq of reporter libraries total RNA with reporter mRNA libraries was ligated to a pre adenylated 3ʹ adapter directly in most cases but for the N60 library injected into fish embryos and frog oocytes the N37 PAS N17 library injected into fish embryos and frog oocytes and the CPEmos N60 and N60LC PASmos libraries injected into frog oocytes reporter library mRNAs were enriched by anti sense oligo capture with biotinylated oligos. RNA isolated from the oocyte or embryo lysate was mixed with 8 pmol KXSH009 8 pmol KXSH010 and 2x SSC 0.3 M NaCl 30 mM sodium citrate pH 7.0 in a total of 50 µl. The RNA and oligos were annealed by incubation at 70°C for 5 min and then slowly cooling to 23°C at 0.1°C/sec. The annealed mixture was combined with 40 µl MyOne Streptavidin C1 beads Thermo Fisher 65002 and incubated for 20 min at 23°C on a thermal mixer shaking with 15 sec on and 1 min 45 sec off. The superna…   tissue:embryo|treatment:N1 GSM7716868 GSM7716868: Fish embryo mRNA 1024cell PAL seq v4; Danio rerio; OTHER GSM7716868 r1 GSM7716868 1 Frog oocytes and embryos were lysed in ice cold buffer RL 20 mM HEPES pH 7.5 100 mM KCl 5 mM MgCl2 1% [v/v] Triton X 100 100 µg/ml cycloheximide cOmplete protease inhibitor cocktail [1 tablet per 10 ml buffer] and 200 units/ml SUPERase•In in a volume of 10 µl per oocyte/embryo by vigorous shaking and pipetting. Lysates were cleared by centrifugation at 5000 g at 4°C for 10 min. The supernatant was transfered to a new tube and mixed with the Tri reagent for RNA isolation. Fish embryos were de chorionated by incubation with 2 mg/ml pronase in E3 medium for 4 min. post removing all E3 medium Tri Reagent was added to the embryos for RNA isolation. Mouse GV oocytes were collected in 37°C MEM in the presence of milrinone to prevent maturation. Cumulus cells were removed from cumulus oocyte complexes by repeated aspiration through a glass pipette. GV oocytes were then collected in TRI reagent for RNA isolation. Mouse MII oocytes were harvested from the oviducts of hCG induced mice 16 hr denuded with 3 mg/ml hyaluronidase for 2 min washed and then collected in Tri reagent for RNA isolation. For gene specific tail seq of reporter libraries total RNA with reporter mRNA libraries was ligated to a pre adenylated 3ʹ adapter directly in most cases but for the N60 library injected into fish embryos and frog oocytes the N37 PAS N17 library injected into fish embryos and frog oocytes and the CPEmos N60 and N60LC PASmos libraries injected into frog oocytes reporter library mRNAs were enriched by anti sense oligo capture with biotinylated oligos. RNA isolated from the oocyte or embryo lysate was mixed with 8 pmol KXSH009 8 pmol KXSH010 and 2x SSC 0.3 M NaCl 30 mM sodium citrate pH 7.0 in a total of 50 µl. The RNA and oligos were annealed by incubation at 70°C for 5 min and then slowly cooling to 23°C at 0.1°C/sec. The annealed mixture was combined with 40 µl MyOne Streptavidin C1 beads Thermo Fisher 65002 and incubated for 20 min at 23°C on a thermal mixer shaking with 15 sec on and 1 min 45 sec off. The superna…   OTHER TRANSCRIPTOMIC other PAIRED ILLUMINA Illumina HiSeq 2500   SRP455680     Fish_embryo_mRNA_1024cell_PAL_seq_v4_rep1_raw_read1.fastq.gz Fish_embryo_mRNA_1024cell_PAL_seq_v4_rep1_raw_read2.fastq.gz fastq fastq 2191900794.0 7139742.0 GSM7716868 r1 0:52 1:255 A:571954354;C:551162617;G:572445400;T:490238669;N:6099754 52 255     571954354 551162617 572445400 490238669 6099754 SRX21396039 SRS18636197 SRA1694849 Whitehead Institute Whitehead Institute 2 0.0011 0.43387 6e-05 0.01058 0.99864 0.99971 0.74576 1.0 52 255 T B mate1 technical by mapping diff illumina hiseq_era unknown poly_a unknown bulk bulk bulk   United States 2023-08-17 Zygote Embryo Embryo Imprecise All anatomical structures
25189 25189 SRR25670736 SRX21396039 SRS18636197 SRP455680 PRJNA1006406 Control of polyA tail length and translation in vertebrate oocytes and early embryos GSE241107 Other During oocyte maturation and early embryonic development polyA tail lengths strongly influence mRNA translation. However how tail lengths are controlled at different developmental stages has been unclear. Here we performed tail length and translational profiling of mRNA reporter libraries each with > 10 million three prime UTR sequence variants in frog oocytes and embryos and fish embryos. These analyses revealed that the UUUUA motif specifies cytoplasmic polyadenylation and identified diverse context features that modulate the activity of this 5 mer. Additional sequence motifs drive stage specific deadenylation in embryos and UUUUA and C rich motifs drive tail length independent translational repression in oocytes. A neural network model accurately predicts tail length change during oocyte maturation in frogs mice and humans. Analyses of human sequence variants showed that those predicted to disrupt tail length control have been under negative selection implying that our insights into control of polyA tail length and translation have implications for human health and fertility. Overall design: Sythetic mRNA reporter libraries with random three prime UTR sequences under four different sequence contexts were injected into frog oocytes and embryos and fish embryos. PolyA tail lengths were measured to at different developmental stages to examine sequence motifs that caused tail length changes. At the same time polyA tail lengths of endogenous mRNAs from frog oocytes and embryos fish embryos and mouse oocytes were measured to investiage how their tail lengths were controlled. Please note that sample titles have been updated on Jan 6 2024.       Fish embryo mRNA 1024cell PAL seq v4 GSM7716868   source name:embryo|tissue:embryo|treatment:N1|geo loc name:missing|collection date:missing Fish embryo mRNA 1024cell PAL seq v4 For gene specific tail seq of mRNA reporters data were processed with a custom script available at https://github.com/coffeebond/MPRA tail seq. For PAL seq v3 or v4 reads were trimmed with cutadapt v3.7 with the parameters “ m 15 quality base=64 q 20 20 match read wildcards e 0.05 a NNNNATCTCGTATGCCGTCTTCTGCTTG O 7”. The trimmed reads were mapped using STAR v2.7.1a to the reference database containing the genomic sequences of the organism from which the mRNAs were obtained the genomic sequences of humans or fish depending on which spike in RNAs were used and the sequences of the polyA standards generated previously with the parameters “ runThreadN 16 runMode alignReads outFilterMultimapNmax 1 outReadsUnmapped Fastx outFilterType BySJout outSAMattributes All outSAMtype BAM Unsorted SortedByCoordinate”. Uniquely mapped read were furhter processed with a custom script available at https://github.com/coffeebond/PAL seq. Assembly: Homo sapiens: GRCh38.p7 primary assembly. Mus muculus: GRCh38.p4 primary assembly. Xenopus laevis: v10.1 assembly. Danio rerio: GRCz11 assembly. Supplementary files format and content: For gene specific tail seq of mRNA reporters tab delimited text files with columns indicating 1 sequence of the variable region; 2 median tail length; 3 number of reads; 4–7 tail lengths at quantile 10 25 75 and 90. Supplementary files format and content: For PAL seq v3 or v4 tab delimited files with columns indicating 1 gene ID or polyA site ID; 2 read cluster ID; 3 tail length Library strategy: PAL seq v4 embryo   Frog oocytes and embryos were lysed in ice cold buffer RL 20 mM HEPES pH 7.5 100 mM KCl 5 mM MgCl2 1% [v/v] Triton X 100 100 µg/ml cycloheximide cOmplete protease inhibitor cocktail [1 tablet per 10 ml buffer] and 200 units/ml SUPERase•In in a volume of 10 µl per oocyte/embryo by vigorous shaking and pipetting. Lysates were cleared by centrifugation at 5000 g at 4°C for 10 min. The supernatant was transfered to a new tube and mixed with the Tri reagent for RNA isolation. Fish embryos were de chorionated by incubation with 2 mg/ml pronase in E3 medium for 4 min. post removing all E3 medium Tri Reagent was added to the embryos for RNA isolation. Mouse GV oocytes were collected in 37°C MEM in the presence of milrinone to prevent maturation. Cumulus cells were removed from cumulus oocyte complexes by repeated aspiration through a glass pipette. GV oocytes were then collected in TRI reagent for RNA isolation. Mouse MII oocytes were harvested from the oviducts of hCG induced mice 16 hr denuded with 3 mg/ml hyaluronidase for 2 min washed and then collected in Tri reagent for RNA isolation. For gene specific tail seq of reporter libraries total RNA with reporter mRNA libraries was ligated to a pre adenylated 3ʹ adapter directly in most cases but for the N60 library injected into fish embryos and frog oocytes the N37 PAS N17 library injected into fish embryos and frog oocytes and the CPEmos N60 and N60LC PASmos libraries injected into frog oocytes reporter library mRNAs were enriched by anti sense oligo capture with biotinylated oligos. RNA isolated from the oocyte or embryo lysate was mixed with 8 pmol KXSH009 8 pmol KXSH010 and 2x SSC 0.3 M NaCl 30 mM sodium citrate pH 7.0 in a total of 50 µl. The RNA and oligos were annealed by incubation at 70°C for 5 min and then slowly cooling to 23°C at 0.1°C/sec. The annealed mixture was combined with 40 µl MyOne Streptavidin C1 beads Thermo Fisher 65002 and incubated for 20 min at 23°C on a thermal mixer shaking with 15 sec on and 1 min 45 sec off. The superna…   tissue:embryo|treatment:N1 GSM7716868 GSM7716868: Fish embryo mRNA 1024cell PAL seq v4; Danio rerio; OTHER GSM7716868 r1 GSM7716868 1 Frog oocytes and embryos were lysed in ice cold buffer RL 20 mM HEPES pH 7.5 100 mM KCl 5 mM MgCl2 1% [v/v] Triton X 100 100 µg/ml cycloheximide cOmplete protease inhibitor cocktail [1 tablet per 10 ml buffer] and 200 units/ml SUPERase•In in a volume of 10 µl per oocyte/embryo by vigorous shaking and pipetting. Lysates were cleared by centrifugation at 5000 g at 4°C for 10 min. The supernatant was transfered to a new tube and mixed with the Tri reagent for RNA isolation. Fish embryos were de chorionated by incubation with 2 mg/ml pronase in E3 medium for 4 min. post removing all E3 medium Tri Reagent was added to the embryos for RNA isolation. Mouse GV oocytes were collected in 37°C MEM in the presence of milrinone to prevent maturation. Cumulus cells were removed from cumulus oocyte complexes by repeated aspiration through a glass pipette. GV oocytes were then collected in TRI reagent for RNA isolation. Mouse MII oocytes were harvested from the oviducts of hCG induced mice 16 hr denuded with 3 mg/ml hyaluronidase for 2 min washed and then collected in Tri reagent for RNA isolation. For gene specific tail seq of reporter libraries total RNA with reporter mRNA libraries was ligated to a pre adenylated 3ʹ adapter directly in most cases but for the N60 library injected into fish embryos and frog oocytes the N37 PAS N17 library injected into fish embryos and frog oocytes and the CPEmos N60 and N60LC PASmos libraries injected into frog oocytes reporter library mRNAs were enriched by anti sense oligo capture with biotinylated oligos. RNA isolated from the oocyte or embryo lysate was mixed with 8 pmol KXSH009 8 pmol KXSH010 and 2x SSC 0.3 M NaCl 30 mM sodium citrate pH 7.0 in a total of 50 µl. The RNA and oligos were annealed by incubation at 70°C for 5 min and then slowly cooling to 23°C at 0.1°C/sec. The annealed mixture was combined with 40 µl MyOne Streptavidin C1 beads Thermo Fisher 65002 and incubated for 20 min at 23°C on a thermal mixer shaking with 15 sec on and 1 min 45 sec off. The superna…   OTHER TRANSCRIPTOMIC other PAIRED ILLUMINA Illumina HiSeq 2500   SRP455680     Fish_embryo_mRNA_1024cell_PAL_seq_v4_rep2_raw_read1.fastq.gz Fish_embryo_mRNA_1024cell_PAL_seq_v4_rep2_raw_read2.fastq.gz fastq fastq 3840723193.0 12510499.0 GSM7716868 r2 0:52 1:255 A:985340216;C:991311706;G:1034487140;T:821820974;N:7763157 52 255     985340216 991311706 1034487140 821820974 7763157 SRX21396039 SRS18636197 SRA1694849 Whitehead Institute Whitehead Institute 2 0.0021 0.0 0.00026 0.0 0.99859 1.0 0.71022   52 255 T T mates < 9% mapping rate illumina hiseq_era unknown poly_a unknown bulk bulk bulk   United States 2023-08-17 Zygote Embryo Embryo Imprecise All anatomical structures
25190 25190 SRR25670737 SRX21396038 SRS18636196 SRP455680 PRJNA1006406 Control of polyA tail length and translation in vertebrate oocytes and early embryos GSE241107 Other During oocyte maturation and early embryonic development polyA tail lengths strongly influence mRNA translation. However how tail lengths are controlled at different developmental stages has been unclear. Here we performed tail length and translational profiling of mRNA reporter libraries each with > 10 million three prime UTR sequence variants in frog oocytes and embryos and fish embryos. These analyses revealed that the UUUUA motif specifies cytoplasmic polyadenylation and identified diverse context features that modulate the activity of this 5 mer. Additional sequence motifs drive stage specific deadenylation in embryos and UUUUA and C rich motifs drive tail length independent translational repression in oocytes. A neural network model accurately predicts tail length change during oocyte maturation in frogs mice and humans. Analyses of human sequence variants showed that those predicted to disrupt tail length control have been under negative selection implying that our insights into control of polyA tail length and translation have implications for human health and fertility. Overall design: Sythetic mRNA reporter libraries with random three prime UTR sequences under four different sequence contexts were injected into frog oocytes and embryos and fish embryos. PolyA tail lengths were measured to at different developmental stages to examine sequence motifs that caused tail length changes. At the same time polyA tail lengths of endogenous mRNAs from frog oocytes and embryos fish embryos and mouse oocytes were measured to investiage how their tail lengths were controlled. Please note that sample titles have been updated on Jan 6 2024.       Fish embryo mRNA 128cell PAL seq v4 GSM7716867   source name:embryo|tissue:embryo|treatment:N1|geo loc name:missing|collection date:missing Fish embryo mRNA 128cell PAL seq v4 For gene specific tail seq of mRNA reporters data were processed with a custom script available at https://github.com/coffeebond/MPRA tail seq. For PAL seq v3 or v4 reads were trimmed with cutadapt v3.7 with the parameters “ m 15 quality base=64 q 20 20 match read wildcards e 0.05 a NNNNATCTCGTATGCCGTCTTCTGCTTG O 7”. The trimmed reads were mapped using STAR v2.7.1a to the reference database containing the genomic sequences of the organism from which the mRNAs were obtained the genomic sequences of humans or fish depending on which spike in RNAs were used and the sequences of the polyA standards generated previously with the parameters “ runThreadN 16 runMode alignReads outFilterMultimapNmax 1 outReadsUnmapped Fastx outFilterType BySJout outSAMattributes All outSAMtype BAM Unsorted SortedByCoordinate”. Uniquely mapped read were furhter processed with a custom script available at https://github.com/coffeebond/PAL seq. Assembly: Homo sapiens: GRCh38.p7 primary assembly. Mus muculus: GRCh38.p4 primary assembly. Xenopus laevis: v10.1 assembly. Danio rerio: GRCz11 assembly. Supplementary files format and content: For gene specific tail seq of mRNA reporters tab delimited text files with columns indicating 1 sequence of the variable region; 2 median tail length; 3 number of reads; 4–7 tail lengths at quantile 10 25 75 and 90. Supplementary files format and content: For PAL seq v3 or v4 tab delimited files with columns indicating 1 gene ID or polyA site ID; 2 read cluster ID; 3 tail length Library strategy: PAL seq v4 embryo   Frog oocytes and embryos were lysed in ice cold buffer RL 20 mM HEPES pH 7.5 100 mM KCl 5 mM MgCl2 1% [v/v] Triton X 100 100 µg/ml cycloheximide cOmplete protease inhibitor cocktail [1 tablet per 10 ml buffer] and 200 units/ml SUPERase•In in a volume of 10 µl per oocyte/embryo by vigorous shaking and pipetting. Lysates were cleared by centrifugation at 5000 g at 4°C for 10 min. The supernatant was transfered to a new tube and mixed with the Tri reagent for RNA isolation. Fish embryos were de chorionated by incubation with 2 mg/ml pronase in E3 medium for 4 min. post removing all E3 medium Tri Reagent was added to the embryos for RNA isolation. Mouse GV oocytes were collected in 37°C MEM in the presence of milrinone to prevent maturation. Cumulus cells were removed from cumulus oocyte complexes by repeated aspiration through a glass pipette. GV oocytes were then collected in TRI reagent for RNA isolation. Mouse MII oocytes were harvested from the oviducts of hCG induced mice 16 hr denuded with 3 mg/ml hyaluronidase for 2 min washed and then collected in Tri reagent for RNA isolation. For gene specific tail seq of reporter libraries total RNA with reporter mRNA libraries was ligated to a pre adenylated 3ʹ adapter directly in most cases but for the N60 library injected into fish embryos and frog oocytes the N37 PAS N17 library injected into fish embryos and frog oocytes and the CPEmos N60 and N60LC PASmos libraries injected into frog oocytes reporter library mRNAs were enriched by anti sense oligo capture with biotinylated oligos. RNA isolated from the oocyte or embryo lysate was mixed with 8 pmol KXSH009 8 pmol KXSH010 and 2x SSC 0.3 M NaCl 30 mM sodium citrate pH 7.0 in a total of 50 µl. The RNA and oligos were annealed by incubation at 70°C for 5 min and then slowly cooling to 23°C at 0.1°C/sec. The annealed mixture was combined with 40 µl MyOne Streptavidin C1 beads Thermo Fisher 65002 and incubated for 20 min at 23°C on a thermal mixer shaking with 15 sec on and 1 min 45 sec off. The superna…   tissue:embryo|treatment:N1 GSM7716867 GSM7716867: Fish embryo mRNA 128cell PAL seq v4; Danio rerio; OTHER GSM7716867 r1 GSM7716867 1 Frog oocytes and embryos were lysed in ice cold buffer RL 20 mM HEPES pH 7.5 100 mM KCl 5 mM MgCl2 1% [v/v] Triton X 100 100 µg/ml cycloheximide cOmplete protease inhibitor cocktail [1 tablet per 10 ml buffer] and 200 units/ml SUPERase•In in a volume of 10 µl per oocyte/embryo by vigorous shaking and pipetting. Lysates were cleared by centrifugation at 5000 g at 4°C for 10 min. The supernatant was transfered to a new tube and mixed with the Tri reagent for RNA isolation. Fish embryos were de chorionated by incubation with 2 mg/ml pronase in E3 medium for 4 min. post removing all E3 medium Tri Reagent was added to the embryos for RNA isolation. Mouse GV oocytes were collected in 37°C MEM in the presence of milrinone to prevent maturation. Cumulus cells were removed from cumulus oocyte complexes by repeated aspiration through a glass pipette. GV oocytes were then collected in TRI reagent for RNA isolation. Mouse MII oocytes were harvested from the oviducts of hCG induced mice 16 hr denuded with 3 mg/ml hyaluronidase for 2 min washed and then collected in Tri reagent for RNA isolation. For gene specific tail seq of reporter libraries total RNA with reporter mRNA libraries was ligated to a pre adenylated 3ʹ adapter directly in most cases but for the N60 library injected into fish embryos and frog oocytes the N37 PAS N17 library injected into fish embryos and frog oocytes and the CPEmos N60 and N60LC PASmos libraries injected into frog oocytes reporter library mRNAs were enriched by anti sense oligo capture with biotinylated oligos. RNA isolated from the oocyte or embryo lysate was mixed with 8 pmol KXSH009 8 pmol KXSH010 and 2x SSC 0.3 M NaCl 30 mM sodium citrate pH 7.0 in a total of 50 µl. The RNA and oligos were annealed by incubation at 70°C for 5 min and then slowly cooling to 23°C at 0.1°C/sec. The annealed mixture was combined with 40 µl MyOne Streptavidin C1 beads Thermo Fisher 65002 and incubated for 20 min at 23°C on a thermal mixer shaking with 15 sec on and 1 min 45 sec off. The superna…   OTHER TRANSCRIPTOMIC other PAIRED ILLUMINA Illumina HiSeq 2500   SRP455680     Fish_embryo_mRNA_128cell_PAL_seq_v4_rep1_raw_read1.fastq.gz Fish_embryo_mRNA_128cell_PAL_seq_v4_rep1_raw_read2.fastq.gz fastq fastq 2104868443.0 6856249.0 GSM7716867 r1 0:52 1:255 A:539122676;C:505234642;G:558793048;T:495876292;N:5841785 52 255     539122676 505234642 558793048 495876292 5841785 SRX21396038 SRS18636196 SRA1694849 Whitehead Institute Whitehead Institute 2 0.00035 0.29379 7e-05 0.01129 0.99949 0.99971 0.55882 0.79591 52 255 T B mate1 technical by mapping diff illumina hiseq_era unknown poly_a unknown bulk bulk bulk   United States 2023-08-17 Multi-stage Embryo Embryo Imprecise All anatomical structures
25191 25191 SRR25670738 SRX21396038 SRS18636196 SRP455680 PRJNA1006406 Control of polyA tail length and translation in vertebrate oocytes and early embryos GSE241107 Other During oocyte maturation and early embryonic development polyA tail lengths strongly influence mRNA translation. However how tail lengths are controlled at different developmental stages has been unclear. Here we performed tail length and translational profiling of mRNA reporter libraries each with > 10 million three prime UTR sequence variants in frog oocytes and embryos and fish embryos. These analyses revealed that the UUUUA motif specifies cytoplasmic polyadenylation and identified diverse context features that modulate the activity of this 5 mer. Additional sequence motifs drive stage specific deadenylation in embryos and UUUUA and C rich motifs drive tail length independent translational repression in oocytes. A neural network model accurately predicts tail length change during oocyte maturation in frogs mice and humans. Analyses of human sequence variants showed that those predicted to disrupt tail length control have been under negative selection implying that our insights into control of polyA tail length and translation have implications for human health and fertility. Overall design: Sythetic mRNA reporter libraries with random three prime UTR sequences under four different sequence contexts were injected into frog oocytes and embryos and fish embryos. PolyA tail lengths were measured to at different developmental stages to examine sequence motifs that caused tail length changes. At the same time polyA tail lengths of endogenous mRNAs from frog oocytes and embryos fish embryos and mouse oocytes were measured to investiage how their tail lengths were controlled. Please note that sample titles have been updated on Jan 6 2024.       Fish embryo mRNA 128cell PAL seq v4 GSM7716867   source name:embryo|tissue:embryo|treatment:N1|geo loc name:missing|collection date:missing Fish embryo mRNA 128cell PAL seq v4 For gene specific tail seq of mRNA reporters data were processed with a custom script available at https://github.com/coffeebond/MPRA tail seq. For PAL seq v3 or v4 reads were trimmed with cutadapt v3.7 with the parameters “ m 15 quality base=64 q 20 20 match read wildcards e 0.05 a NNNNATCTCGTATGCCGTCTTCTGCTTG O 7”. The trimmed reads were mapped using STAR v2.7.1a to the reference database containing the genomic sequences of the organism from which the mRNAs were obtained the genomic sequences of humans or fish depending on which spike in RNAs were used and the sequences of the polyA standards generated previously with the parameters “ runThreadN 16 runMode alignReads outFilterMultimapNmax 1 outReadsUnmapped Fastx outFilterType BySJout outSAMattributes All outSAMtype BAM Unsorted SortedByCoordinate”. Uniquely mapped read were furhter processed with a custom script available at https://github.com/coffeebond/PAL seq. Assembly: Homo sapiens: GRCh38.p7 primary assembly. Mus muculus: GRCh38.p4 primary assembly. Xenopus laevis: v10.1 assembly. Danio rerio: GRCz11 assembly. Supplementary files format and content: For gene specific tail seq of mRNA reporters tab delimited text files with columns indicating 1 sequence of the variable region; 2 median tail length; 3 number of reads; 4–7 tail lengths at quantile 10 25 75 and 90. Supplementary files format and content: For PAL seq v3 or v4 tab delimited files with columns indicating 1 gene ID or polyA site ID; 2 read cluster ID; 3 tail length Library strategy: PAL seq v4 embryo   Frog oocytes and embryos were lysed in ice cold buffer RL 20 mM HEPES pH 7.5 100 mM KCl 5 mM MgCl2 1% [v/v] Triton X 100 100 µg/ml cycloheximide cOmplete protease inhibitor cocktail [1 tablet per 10 ml buffer] and 200 units/ml SUPERase•In in a volume of 10 µl per oocyte/embryo by vigorous shaking and pipetting. Lysates were cleared by centrifugation at 5000 g at 4°C for 10 min. The supernatant was transfered to a new tube and mixed with the Tri reagent for RNA isolation. Fish embryos were de chorionated by incubation with 2 mg/ml pronase in E3 medium for 4 min. post removing all E3 medium Tri Reagent was added to the embryos for RNA isolation. Mouse GV oocytes were collected in 37°C MEM in the presence of milrinone to prevent maturation. Cumulus cells were removed from cumulus oocyte complexes by repeated aspiration through a glass pipette. GV oocytes were then collected in TRI reagent for RNA isolation. Mouse MII oocytes were harvested from the oviducts of hCG induced mice 16 hr denuded with 3 mg/ml hyaluronidase for 2 min washed and then collected in Tri reagent for RNA isolation. For gene specific tail seq of reporter libraries total RNA with reporter mRNA libraries was ligated to a pre adenylated 3ʹ adapter directly in most cases but for the N60 library injected into fish embryos and frog oocytes the N37 PAS N17 library injected into fish embryos and frog oocytes and the CPEmos N60 and N60LC PASmos libraries injected into frog oocytes reporter library mRNAs were enriched by anti sense oligo capture with biotinylated oligos. RNA isolated from the oocyte or embryo lysate was mixed with 8 pmol KXSH009 8 pmol KXSH010 and 2x SSC 0.3 M NaCl 30 mM sodium citrate pH 7.0 in a total of 50 µl. The RNA and oligos were annealed by incubation at 70°C for 5 min and then slowly cooling to 23°C at 0.1°C/sec. The annealed mixture was combined with 40 µl MyOne Streptavidin C1 beads Thermo Fisher 65002 and incubated for 20 min at 23°C on a thermal mixer shaking with 15 sec on and 1 min 45 sec off. The superna…   tissue:embryo|treatment:N1 GSM7716867 GSM7716867: Fish embryo mRNA 128cell PAL seq v4; Danio rerio; OTHER GSM7716867 r1 GSM7716867 1 Frog oocytes and embryos were lysed in ice cold buffer RL 20 mM HEPES pH 7.5 100 mM KCl 5 mM MgCl2 1% [v/v] Triton X 100 100 µg/ml cycloheximide cOmplete protease inhibitor cocktail [1 tablet per 10 ml buffer] and 200 units/ml SUPERase•In in a volume of 10 µl per oocyte/embryo by vigorous shaking and pipetting. Lysates were cleared by centrifugation at 5000 g at 4°C for 10 min. The supernatant was transfered to a new tube and mixed with the Tri reagent for RNA isolation. Fish embryos were de chorionated by incubation with 2 mg/ml pronase in E3 medium for 4 min. post removing all E3 medium Tri Reagent was added to the embryos for RNA isolation. Mouse GV oocytes were collected in 37°C MEM in the presence of milrinone to prevent maturation. Cumulus cells were removed from cumulus oocyte complexes by repeated aspiration through a glass pipette. GV oocytes were then collected in TRI reagent for RNA isolation. Mouse MII oocytes were harvested from the oviducts of hCG induced mice 16 hr denuded with 3 mg/ml hyaluronidase for 2 min washed and then collected in Tri reagent for RNA isolation. For gene specific tail seq of reporter libraries total RNA with reporter mRNA libraries was ligated to a pre adenylated 3ʹ adapter directly in most cases but for the N60 library injected into fish embryos and frog oocytes the N37 PAS N17 library injected into fish embryos and frog oocytes and the CPEmos N60 and N60LC PASmos libraries injected into frog oocytes reporter library mRNAs were enriched by anti sense oligo capture with biotinylated oligos. RNA isolated from the oocyte or embryo lysate was mixed with 8 pmol KXSH009 8 pmol KXSH010 and 2x SSC 0.3 M NaCl 30 mM sodium citrate pH 7.0 in a total of 50 µl. The RNA and oligos were annealed by incubation at 70°C for 5 min and then slowly cooling to 23°C at 0.1°C/sec. The annealed mixture was combined with 40 µl MyOne Streptavidin C1 beads Thermo Fisher 65002 and incubated for 20 min at 23°C on a thermal mixer shaking with 15 sec on and 1 min 45 sec off. The superna…   OTHER TRANSCRIPTOMIC other PAIRED ILLUMINA Illumina HiSeq 2500   SRP455680     Fish_embryo_mRNA_128cell_PAL_seq_v4_rep2_raw_read1.fastq.gz Fish_embryo_mRNA_128cell_PAL_seq_v4_rep2_raw_read2.fastq.gz fastq fastq 3135806371.0 10214353.0 GSM7716867 r2 0:52 1:255 A:776612486;C:774126305;G:853951502;T:724788612;N:6327466 52 255     776612486 774126305 853951502 724788612 6327466 SRX21396038 SRS18636196 SRA1694849 Whitehead Institute Whitehead Institute 2 0.00066 0.0 0.00011 0.0 0.99939 1.0 0.55737   52 255 T T mates < 9% mapping rate illumina hiseq_era unknown poly_a unknown bulk bulk bulk   United States 2023-08-17 Multi-stage Embryo Embryo Imprecise All anatomical structures
25192 25192 SRR25670739 SRX21396037 SRS18636195 SRP455680 PRJNA1006406 Control of polyA tail length and translation in vertebrate oocytes and early embryos GSE241107 Other During oocyte maturation and early embryonic development polyA tail lengths strongly influence mRNA translation. However how tail lengths are controlled at different developmental stages has been unclear. Here we performed tail length and translational profiling of mRNA reporter libraries each with > 10 million three prime UTR sequence variants in frog oocytes and embryos and fish embryos. These analyses revealed that the UUUUA motif specifies cytoplasmic polyadenylation and identified diverse context features that modulate the activity of this 5 mer. Additional sequence motifs drive stage specific deadenylation in embryos and UUUUA and C rich motifs drive tail length independent translational repression in oocytes. A neural network model accurately predicts tail length change during oocyte maturation in frogs mice and humans. Analyses of human sequence variants showed that those predicted to disrupt tail length control have been under negative selection implying that our insights into control of polyA tail length and translation have implications for human health and fertility. Overall design: Sythetic mRNA reporter libraries with random three prime UTR sequences under four different sequence contexts were injected into frog oocytes and embryos and fish embryos. PolyA tail lengths were measured to at different developmental stages to examine sequence motifs that caused tail length changes. At the same time polyA tail lengths of endogenous mRNAs from frog oocytes and embryos fish embryos and mouse oocytes were measured to investiage how their tail lengths were controlled. Please note that sample titles have been updated on Jan 6 2024.       Fish embryo mRNA 8cell PAL seq v4 GSM7716866   source name:embryo|tissue:embryo|treatment:N1|geo loc name:missing|collection date:missing Fish embryo mRNA 8cell PAL seq v4 For gene specific tail seq of mRNA reporters data were processed with a custom script available at https://github.com/coffeebond/MPRA tail seq. For PAL seq v3 or v4 reads were trimmed with cutadapt v3.7 with the parameters “ m 15 quality base=64 q 20 20 match read wildcards e 0.05 a NNNNATCTCGTATGCCGTCTTCTGCTTG O 7”. The trimmed reads were mapped using STAR v2.7.1a to the reference database containing the genomic sequences of the organism from which the mRNAs were obtained the genomic sequences of humans or fish depending on which spike in RNAs were used and the sequences of the polyA standards generated previously with the parameters “ runThreadN 16 runMode alignReads outFilterMultimapNmax 1 outReadsUnmapped Fastx outFilterType BySJout outSAMattributes All outSAMtype BAM Unsorted SortedByCoordinate”. Uniquely mapped read were furhter processed with a custom script available at https://github.com/coffeebond/PAL seq. Assembly: Homo sapiens: GRCh38.p7 primary assembly. Mus muculus: GRCh38.p4 primary assembly. Xenopus laevis: v10.1 assembly. Danio rerio: GRCz11 assembly. Supplementary files format and content: For gene specific tail seq of mRNA reporters tab delimited text files with columns indicating 1 sequence of the variable region; 2 median tail length; 3 number of reads; 4–7 tail lengths at quantile 10 25 75 and 90. Supplementary files format and content: For PAL seq v3 or v4 tab delimited files with columns indicating 1 gene ID or polyA site ID; 2 read cluster ID; 3 tail length Library strategy: PAL seq v4 embryo   Frog oocytes and embryos were lysed in ice cold buffer RL 20 mM HEPES pH 7.5 100 mM KCl 5 mM MgCl2 1% [v/v] Triton X 100 100 µg/ml cycloheximide cOmplete protease inhibitor cocktail [1 tablet per 10 ml buffer] and 200 units/ml SUPERase•In in a volume of 10 µl per oocyte/embryo by vigorous shaking and pipetting. Lysates were cleared by centrifugation at 5000 g at 4°C for 10 min. The supernatant was transfered to a new tube and mixed with the Tri reagent for RNA isolation. Fish embryos were de chorionated by incubation with 2 mg/ml pronase in E3 medium for 4 min. post removing all E3 medium Tri Reagent was added to the embryos for RNA isolation. Mouse GV oocytes were collected in 37°C MEM in the presence of milrinone to prevent maturation. Cumulus cells were removed from cumulus oocyte complexes by repeated aspiration through a glass pipette. GV oocytes were then collected in TRI reagent for RNA isolation. Mouse MII oocytes were harvested from the oviducts of hCG induced mice 16 hr denuded with 3 mg/ml hyaluronidase for 2 min washed and then collected in Tri reagent for RNA isolation. For gene specific tail seq of reporter libraries total RNA with reporter mRNA libraries was ligated to a pre adenylated 3ʹ adapter directly in most cases but for the N60 library injected into fish embryos and frog oocytes the N37 PAS N17 library injected into fish embryos and frog oocytes and the CPEmos N60 and N60LC PASmos libraries injected into frog oocytes reporter library mRNAs were enriched by anti sense oligo capture with biotinylated oligos. RNA isolated from the oocyte or embryo lysate was mixed with 8 pmol KXSH009 8 pmol KXSH010 and 2x SSC 0.3 M NaCl 30 mM sodium citrate pH 7.0 in a total of 50 µl. The RNA and oligos were annealed by incubation at 70°C for 5 min and then slowly cooling to 23°C at 0.1°C/sec. The annealed mixture was combined with 40 µl MyOne Streptavidin C1 beads Thermo Fisher 65002 and incubated for 20 min at 23°C on a thermal mixer shaking with 15 sec on and 1 min 45 sec off. The superna…   tissue:embryo|treatment:N1 GSM7716866 GSM7716866: Fish embryo mRNA 8cell PAL seq v4; Danio rerio; OTHER GSM7716866 r1 GSM7716866 1 Frog oocytes and embryos were lysed in ice cold buffer RL 20 mM HEPES pH 7.5 100 mM KCl 5 mM MgCl2 1% [v/v] Triton X 100 100 µg/ml cycloheximide cOmplete protease inhibitor cocktail [1 tablet per 10 ml buffer] and 200 units/ml SUPERase•In in a volume of 10 µl per oocyte/embryo by vigorous shaking and pipetting. Lysates were cleared by centrifugation at 5000 g at 4°C for 10 min. The supernatant was transfered to a new tube and mixed with the Tri reagent for RNA isolation. Fish embryos were de chorionated by incubation with 2 mg/ml pronase in E3 medium for 4 min. post removing all E3 medium Tri Reagent was added to the embryos for RNA isolation. Mouse GV oocytes were collected in 37°C MEM in the presence of milrinone to prevent maturation. Cumulus cells were removed from cumulus oocyte complexes by repeated aspiration through a glass pipette. GV oocytes were then collected in TRI reagent for RNA isolation. Mouse MII oocytes were harvested from the oviducts of hCG induced mice 16 hr denuded with 3 mg/ml hyaluronidase for 2 min washed and then collected in Tri reagent for RNA isolation. For gene specific tail seq of reporter libraries total RNA with reporter mRNA libraries was ligated to a pre adenylated 3ʹ adapter directly in most cases but for the N60 library injected into fish embryos and frog oocytes the N37 PAS N17 library injected into fish embryos and frog oocytes and the CPEmos N60 and N60LC PASmos libraries injected into frog oocytes reporter library mRNAs were enriched by anti sense oligo capture with biotinylated oligos. RNA isolated from the oocyte or embryo lysate was mixed with 8 pmol KXSH009 8 pmol KXSH010 and 2x SSC 0.3 M NaCl 30 mM sodium citrate pH 7.0 in a total of 50 µl. The RNA and oligos were annealed by incubation at 70°C for 5 min and then slowly cooling to 23°C at 0.1°C/sec. The annealed mixture was combined with 40 µl MyOne Streptavidin C1 beads Thermo Fisher 65002 and incubated for 20 min at 23°C on a thermal mixer shaking with 15 sec on and 1 min 45 sec off. The superna…   OTHER TRANSCRIPTOMIC other PAIRED ILLUMINA Illumina HiSeq 2500   SRP455680     Fish_embryo_mRNA_8cell_PAL_seq_v4_rep1_raw_read1.fastq.gz Fish_embryo_mRNA_8cell_PAL_seq_v4_rep1_raw_read2.fastq.gz fastq fastq 2618998887.0 8530941.0 GSM7716866 r1 0:52 1:255 A:673066508;C:641446717;G:706546263;T:590452494;N:7486905 52 255     673066508 641446717 706546263 590452494 7486905 SRX21396037 SRS18636195 SRA1694849 Whitehead Institute Whitehead Institute 2 0.0009 0.43244 0.00014 0.0054 0.99902 0.99967 0.54901 1.0 52 255 T B mate1 technical by mapping diff illumina hiseq_era unknown poly_a unknown bulk bulk bulk   United States 2023-08-17 Multi-stage Embryo Embryo Imprecise All anatomical structures
25193 25193 SRR25670740 SRX21396037 SRS18636195 SRP455680 PRJNA1006406 Control of polyA tail length and translation in vertebrate oocytes and early embryos GSE241107 Other During oocyte maturation and early embryonic development polyA tail lengths strongly influence mRNA translation. However how tail lengths are controlled at different developmental stages has been unclear. Here we performed tail length and translational profiling of mRNA reporter libraries each with > 10 million three prime UTR sequence variants in frog oocytes and embryos and fish embryos. These analyses revealed that the UUUUA motif specifies cytoplasmic polyadenylation and identified diverse context features that modulate the activity of this 5 mer. Additional sequence motifs drive stage specific deadenylation in embryos and UUUUA and C rich motifs drive tail length independent translational repression in oocytes. A neural network model accurately predicts tail length change during oocyte maturation in frogs mice and humans. Analyses of human sequence variants showed that those predicted to disrupt tail length control have been under negative selection implying that our insights into control of polyA tail length and translation have implications for human health and fertility. Overall design: Sythetic mRNA reporter libraries with random three prime UTR sequences under four different sequence contexts were injected into frog oocytes and embryos and fish embryos. PolyA tail lengths were measured to at different developmental stages to examine sequence motifs that caused tail length changes. At the same time polyA tail lengths of endogenous mRNAs from frog oocytes and embryos fish embryos and mouse oocytes were measured to investiage how their tail lengths were controlled. Please note that sample titles have been updated on Jan 6 2024.       Fish embryo mRNA 8cell PAL seq v4 GSM7716866   source name:embryo|tissue:embryo|treatment:N1|geo loc name:missing|collection date:missing Fish embryo mRNA 8cell PAL seq v4 For gene specific tail seq of mRNA reporters data were processed with a custom script available at https://github.com/coffeebond/MPRA tail seq. For PAL seq v3 or v4 reads were trimmed with cutadapt v3.7 with the parameters “ m 15 quality base=64 q 20 20 match read wildcards e 0.05 a NNNNATCTCGTATGCCGTCTTCTGCTTG O 7”. The trimmed reads were mapped using STAR v2.7.1a to the reference database containing the genomic sequences of the organism from which the mRNAs were obtained the genomic sequences of humans or fish depending on which spike in RNAs were used and the sequences of the polyA standards generated previously with the parameters “ runThreadN 16 runMode alignReads outFilterMultimapNmax 1 outReadsUnmapped Fastx outFilterType BySJout outSAMattributes All outSAMtype BAM Unsorted SortedByCoordinate”. Uniquely mapped read were furhter processed with a custom script available at https://github.com/coffeebond/PAL seq. Assembly: Homo sapiens: GRCh38.p7 primary assembly. Mus muculus: GRCh38.p4 primary assembly. Xenopus laevis: v10.1 assembly. Danio rerio: GRCz11 assembly. Supplementary files format and content: For gene specific tail seq of mRNA reporters tab delimited text files with columns indicating 1 sequence of the variable region; 2 median tail length; 3 number of reads; 4–7 tail lengths at quantile 10 25 75 and 90. Supplementary files format and content: For PAL seq v3 or v4 tab delimited files with columns indicating 1 gene ID or polyA site ID; 2 read cluster ID; 3 tail length Library strategy: PAL seq v4 embryo   Frog oocytes and embryos were lysed in ice cold buffer RL 20 mM HEPES pH 7.5 100 mM KCl 5 mM MgCl2 1% [v/v] Triton X 100 100 µg/ml cycloheximide cOmplete protease inhibitor cocktail [1 tablet per 10 ml buffer] and 200 units/ml SUPERase•In in a volume of 10 µl per oocyte/embryo by vigorous shaking and pipetting. Lysates were cleared by centrifugation at 5000 g at 4°C for 10 min. The supernatant was transfered to a new tube and mixed with the Tri reagent for RNA isolation. Fish embryos were de chorionated by incubation with 2 mg/ml pronase in E3 medium for 4 min. post removing all E3 medium Tri Reagent was added to the embryos for RNA isolation. Mouse GV oocytes were collected in 37°C MEM in the presence of milrinone to prevent maturation. Cumulus cells were removed from cumulus oocyte complexes by repeated aspiration through a glass pipette. GV oocytes were then collected in TRI reagent for RNA isolation. Mouse MII oocytes were harvested from the oviducts of hCG induced mice 16 hr denuded with 3 mg/ml hyaluronidase for 2 min washed and then collected in Tri reagent for RNA isolation. For gene specific tail seq of reporter libraries total RNA with reporter mRNA libraries was ligated to a pre adenylated 3ʹ adapter directly in most cases but for the N60 library injected into fish embryos and frog oocytes the N37 PAS N17 library injected into fish embryos and frog oocytes and the CPEmos N60 and N60LC PASmos libraries injected into frog oocytes reporter library mRNAs were enriched by anti sense oligo capture with biotinylated oligos. RNA isolated from the oocyte or embryo lysate was mixed with 8 pmol KXSH009 8 pmol KXSH010 and 2x SSC 0.3 M NaCl 30 mM sodium citrate pH 7.0 in a total of 50 µl. The RNA and oligos were annealed by incubation at 70°C for 5 min and then slowly cooling to 23°C at 0.1°C/sec. The annealed mixture was combined with 40 µl MyOne Streptavidin C1 beads Thermo Fisher 65002 and incubated for 20 min at 23°C on a thermal mixer shaking with 15 sec on and 1 min 45 sec off. The superna…   tissue:embryo|treatment:N1 GSM7716866 GSM7716866: Fish embryo mRNA 8cell PAL seq v4; Danio rerio; OTHER GSM7716866 r1 GSM7716866 1 Frog oocytes and embryos were lysed in ice cold buffer RL 20 mM HEPES pH 7.5 100 mM KCl 5 mM MgCl2 1% [v/v] Triton X 100 100 µg/ml cycloheximide cOmplete protease inhibitor cocktail [1 tablet per 10 ml buffer] and 200 units/ml SUPERase•In in a volume of 10 µl per oocyte/embryo by vigorous shaking and pipetting. Lysates were cleared by centrifugation at 5000 g at 4°C for 10 min. The supernatant was transfered to a new tube and mixed with the Tri reagent for RNA isolation. Fish embryos were de chorionated by incubation with 2 mg/ml pronase in E3 medium for 4 min. post removing all E3 medium Tri Reagent was added to the embryos for RNA isolation. Mouse GV oocytes were collected in 37°C MEM in the presence of milrinone to prevent maturation. Cumulus cells were removed from cumulus oocyte complexes by repeated aspiration through a glass pipette. GV oocytes were then collected in TRI reagent for RNA isolation. Mouse MII oocytes were harvested from the oviducts of hCG induced mice 16 hr denuded with 3 mg/ml hyaluronidase for 2 min washed and then collected in Tri reagent for RNA isolation. For gene specific tail seq of reporter libraries total RNA with reporter mRNA libraries was ligated to a pre adenylated 3ʹ adapter directly in most cases but for the N60 library injected into fish embryos and frog oocytes the N37 PAS N17 library injected into fish embryos and frog oocytes and the CPEmos N60 and N60LC PASmos libraries injected into frog oocytes reporter library mRNAs were enriched by anti sense oligo capture with biotinylated oligos. RNA isolated from the oocyte or embryo lysate was mixed with 8 pmol KXSH009 8 pmol KXSH010 and 2x SSC 0.3 M NaCl 30 mM sodium citrate pH 7.0 in a total of 50 µl. The RNA and oligos were annealed by incubation at 70°C for 5 min and then slowly cooling to 23°C at 0.1°C/sec. The annealed mixture was combined with 40 µl MyOne Streptavidin C1 beads Thermo Fisher 65002 and incubated for 20 min at 23°C on a thermal mixer shaking with 15 sec on and 1 min 45 sec off. The superna…   OTHER TRANSCRIPTOMIC other PAIRED ILLUMINA Illumina HiSeq 2500   SRP455680     Fish_embryo_mRNA_8cell_PAL_seq_v4_rep2_raw_read1.fastq.gz Fish_embryo_mRNA_8cell_PAL_seq_v4_rep2_raw_read2.fastq.gz fastq fastq 3028839589.0 9865927.0 GSM7716866 r2 0:52 1:255 A:756368817;C:758868409;G:836949742;T:670545278;N:6107343 52 255     756368817 758868409 836949742 670545278 6107343 SRX21396037 SRS18636195 SRA1694849 Whitehead Institute Whitehead Institute 2 0.00156 0.0 0.00026 0.0 0.99835 1.0 0.44791   52 255 T T mates < 9% mapping rate illumina hiseq_era unknown poly_a unknown bulk bulk bulk   United States 2023-08-17 Multi-stage Embryo Embryo Imprecise All anatomical structures
25194 25194 SRR25670741 SRX21396036 SRS18636194 SRP455680 PRJNA1006406 Control of polyA tail length and translation in vertebrate oocytes and early embryos GSE241107 Other During oocyte maturation and early embryonic development polyA tail lengths strongly influence mRNA translation. However how tail lengths are controlled at different developmental stages has been unclear. Here we performed tail length and translational profiling of mRNA reporter libraries each with > 10 million three prime UTR sequence variants in frog oocytes and embryos and fish embryos. These analyses revealed that the UUUUA motif specifies cytoplasmic polyadenylation and identified diverse context features that modulate the activity of this 5 mer. Additional sequence motifs drive stage specific deadenylation in embryos and UUUUA and C rich motifs drive tail length independent translational repression in oocytes. A neural network model accurately predicts tail length change during oocyte maturation in frogs mice and humans. Analyses of human sequence variants showed that those predicted to disrupt tail length control have been under negative selection implying that our insights into control of polyA tail length and translation have implications for human health and fertility. Overall design: Sythetic mRNA reporter libraries with random three prime UTR sequences under four different sequence contexts were injected into frog oocytes and embryos and fish embryos. PolyA tail lengths were measured to at different developmental stages to examine sequence motifs that caused tail length changes. At the same time polyA tail lengths of endogenous mRNAs from frog oocytes and embryos fish embryos and mouse oocytes were measured to investiage how their tail lengths were controlled. Please note that sample titles have been updated on Jan 6 2024.       Fish embryo mRNA 1cell PAL seq v4 GSM7716865   source name:embryo|tissue:embryo|treatment:N1|geo loc name:missing|collection date:missing Fish embryo mRNA 1cell PAL seq v4 For gene specific tail seq of mRNA reporters data were processed with a custom script available at https://github.com/coffeebond/MPRA tail seq. For PAL seq v3 or v4 reads were trimmed with cutadapt v3.7 with the parameters “ m 15 quality base=64 q 20 20 match read wildcards e 0.05 a NNNNATCTCGTATGCCGTCTTCTGCTTG O 7”. The trimmed reads were mapped using STAR v2.7.1a to the reference database containing the genomic sequences of the organism from which the mRNAs were obtained the genomic sequences of humans or fish depending on which spike in RNAs were used and the sequences of the polyA standards generated previously with the parameters “ runThreadN 16 runMode alignReads outFilterMultimapNmax 1 outReadsUnmapped Fastx outFilterType BySJout outSAMattributes All outSAMtype BAM Unsorted SortedByCoordinate”. Uniquely mapped read were furhter processed with a custom script available at https://github.com/coffeebond/PAL seq. Assembly: Homo sapiens: GRCh38.p7 primary assembly. Mus muculus: GRCh38.p4 primary assembly. Xenopus laevis: v10.1 assembly. Danio rerio: GRCz11 assembly. Supplementary files format and content: For gene specific tail seq of mRNA reporters tab delimited text files with columns indicating 1 sequence of the variable region; 2 median tail length; 3 number of reads; 4–7 tail lengths at quantile 10 25 75 and 90. Supplementary files format and content: For PAL seq v3 or v4 tab delimited files with columns indicating 1 gene ID or polyA site ID; 2 read cluster ID; 3 tail length Library strategy: PAL seq v4 embryo   Frog oocytes and embryos were lysed in ice cold buffer RL 20 mM HEPES pH 7.5 100 mM KCl 5 mM MgCl2 1% [v/v] Triton X 100 100 µg/ml cycloheximide cOmplete protease inhibitor cocktail [1 tablet per 10 ml buffer] and 200 units/ml SUPERase•In in a volume of 10 µl per oocyte/embryo by vigorous shaking and pipetting. Lysates were cleared by centrifugation at 5000 g at 4°C for 10 min. The supernatant was transfered to a new tube and mixed with the Tri reagent for RNA isolation. Fish embryos were de chorionated by incubation with 2 mg/ml pronase in E3 medium for 4 min. post removing all E3 medium Tri Reagent was added to the embryos for RNA isolation. Mouse GV oocytes were collected in 37°C MEM in the presence of milrinone to prevent maturation. Cumulus cells were removed from cumulus oocyte complexes by repeated aspiration through a glass pipette. GV oocytes were then collected in TRI reagent for RNA isolation. Mouse MII oocytes were harvested from the oviducts of hCG induced mice 16 hr denuded with 3 mg/ml hyaluronidase for 2 min washed and then collected in Tri reagent for RNA isolation. For gene specific tail seq of reporter libraries total RNA with reporter mRNA libraries was ligated to a pre adenylated 3ʹ adapter directly in most cases but for the N60 library injected into fish embryos and frog oocytes the N37 PAS N17 library injected into fish embryos and frog oocytes and the CPEmos N60 and N60LC PASmos libraries injected into frog oocytes reporter library mRNAs were enriched by anti sense oligo capture with biotinylated oligos. RNA isolated from the oocyte or embryo lysate was mixed with 8 pmol KXSH009 8 pmol KXSH010 and 2x SSC 0.3 M NaCl 30 mM sodium citrate pH 7.0 in a total of 50 µl. The RNA and oligos were annealed by incubation at 70°C for 5 min and then slowly cooling to 23°C at 0.1°C/sec. The annealed mixture was combined with 40 µl MyOne Streptavidin C1 beads Thermo Fisher 65002 and incubated for 20 min at 23°C on a thermal mixer shaking with 15 sec on and 1 min 45 sec off. The superna…   tissue:embryo|treatment:N1 GSM7716865 GSM7716865: Fish embryo mRNA 1cell PAL seq v4; Danio rerio; OTHER GSM7716865 r1 GSM7716865 1 Frog oocytes and embryos were lysed in ice cold buffer RL 20 mM HEPES pH 7.5 100 mM KCl 5 mM MgCl2 1% [v/v] Triton X 100 100 µg/ml cycloheximide cOmplete protease inhibitor cocktail [1 tablet per 10 ml buffer] and 200 units/ml SUPERase•In in a volume of 10 µl per oocyte/embryo by vigorous shaking and pipetting. Lysates were cleared by centrifugation at 5000 g at 4°C for 10 min. The supernatant was transfered to a new tube and mixed with the Tri reagent for RNA isolation. Fish embryos were de chorionated by incubation with 2 mg/ml pronase in E3 medium for 4 min. post removing all E3 medium Tri Reagent was added to the embryos for RNA isolation. Mouse GV oocytes were collected in 37°C MEM in the presence of milrinone to prevent maturation. Cumulus cells were removed from cumulus oocyte complexes by repeated aspiration through a glass pipette. GV oocytes were then collected in TRI reagent for RNA isolation. Mouse MII oocytes were harvested from the oviducts of hCG induced mice 16 hr denuded with 3 mg/ml hyaluronidase for 2 min washed and then collected in Tri reagent for RNA isolation. For gene specific tail seq of reporter libraries total RNA with reporter mRNA libraries was ligated to a pre adenylated 3ʹ adapter directly in most cases but for the N60 library injected into fish embryos and frog oocytes the N37 PAS N17 library injected into fish embryos and frog oocytes and the CPEmos N60 and N60LC PASmos libraries injected into frog oocytes reporter library mRNAs were enriched by anti sense oligo capture with biotinylated oligos. RNA isolated from the oocyte or embryo lysate was mixed with 8 pmol KXSH009 8 pmol KXSH010 and 2x SSC 0.3 M NaCl 30 mM sodium citrate pH 7.0 in a total of 50 µl. The RNA and oligos were annealed by incubation at 70°C for 5 min and then slowly cooling to 23°C at 0.1°C/sec. The annealed mixture was combined with 40 µl MyOne Streptavidin C1 beads Thermo Fisher 65002 and incubated for 20 min at 23°C on a thermal mixer shaking with 15 sec on and 1 min 45 sec off. The superna…   OTHER TRANSCRIPTOMIC other PAIRED ILLUMINA Illumina HiSeq 2500   SRP455680     Fish_embryo_mRNA_1cell_PAL_seq_v4_rep1_raw_read1.fastq.gz Fish_embryo_mRNA_1cell_PAL_seq_v4_rep1_raw_read2.fastq.gz fastq fastq 2050143851.0 6677993.0 GSM7716865 r1 0:52 1:255 A:536391564;C:527126186;G:557802929;T:422990166;N:5833006 52 255     536391564 527126186 557802929 422990166 5833006 SRX21396036 SRS18636194 SRA1694849 Whitehead Institute Whitehead Institute 2 0.00016 0.46479 4e-05 0.01408 0.99979 0.99969 0.54545 1.0 52 255 T B mate1 technical by mapping diff illumina hiseq_era unknown poly_a unknown bulk bulk bulk   United States 2023-08-17 Zygote Embryo Embryo Imprecise All anatomical structures
25195 25195 SRR25670742 SRX21396036 SRS18636194 SRP455680 PRJNA1006406 Control of polyA tail length and translation in vertebrate oocytes and early embryos GSE241107 Other During oocyte maturation and early embryonic development polyA tail lengths strongly influence mRNA translation. However how tail lengths are controlled at different developmental stages has been unclear. Here we performed tail length and translational profiling of mRNA reporter libraries each with > 10 million three prime UTR sequence variants in frog oocytes and embryos and fish embryos. These analyses revealed that the UUUUA motif specifies cytoplasmic polyadenylation and identified diverse context features that modulate the activity of this 5 mer. Additional sequence motifs drive stage specific deadenylation in embryos and UUUUA and C rich motifs drive tail length independent translational repression in oocytes. A neural network model accurately predicts tail length change during oocyte maturation in frogs mice and humans. Analyses of human sequence variants showed that those predicted to disrupt tail length control have been under negative selection implying that our insights into control of polyA tail length and translation have implications for human health and fertility. Overall design: Sythetic mRNA reporter libraries with random three prime UTR sequences under four different sequence contexts were injected into frog oocytes and embryos and fish embryos. PolyA tail lengths were measured to at different developmental stages to examine sequence motifs that caused tail length changes. At the same time polyA tail lengths of endogenous mRNAs from frog oocytes and embryos fish embryos and mouse oocytes were measured to investiage how their tail lengths were controlled. Please note that sample titles have been updated on Jan 6 2024.       Fish embryo mRNA 1cell PAL seq v4 GSM7716865   source name:embryo|tissue:embryo|treatment:N1|geo loc name:missing|collection date:missing Fish embryo mRNA 1cell PAL seq v4 For gene specific tail seq of mRNA reporters data were processed with a custom script available at https://github.com/coffeebond/MPRA tail seq. For PAL seq v3 or v4 reads were trimmed with cutadapt v3.7 with the parameters “ m 15 quality base=64 q 20 20 match read wildcards e 0.05 a NNNNATCTCGTATGCCGTCTTCTGCTTG O 7”. The trimmed reads were mapped using STAR v2.7.1a to the reference database containing the genomic sequences of the organism from which the mRNAs were obtained the genomic sequences of humans or fish depending on which spike in RNAs were used and the sequences of the polyA standards generated previously with the parameters “ runThreadN 16 runMode alignReads outFilterMultimapNmax 1 outReadsUnmapped Fastx outFilterType BySJout outSAMattributes All outSAMtype BAM Unsorted SortedByCoordinate”. Uniquely mapped read were furhter processed with a custom script available at https://github.com/coffeebond/PAL seq. Assembly: Homo sapiens: GRCh38.p7 primary assembly. Mus muculus: GRCh38.p4 primary assembly. Xenopus laevis: v10.1 assembly. Danio rerio: GRCz11 assembly. Supplementary files format and content: For gene specific tail seq of mRNA reporters tab delimited text files with columns indicating 1 sequence of the variable region; 2 median tail length; 3 number of reads; 4–7 tail lengths at quantile 10 25 75 and 90. Supplementary files format and content: For PAL seq v3 or v4 tab delimited files with columns indicating 1 gene ID or polyA site ID; 2 read cluster ID; 3 tail length Library strategy: PAL seq v4 embryo   Frog oocytes and embryos were lysed in ice cold buffer RL 20 mM HEPES pH 7.5 100 mM KCl 5 mM MgCl2 1% [v/v] Triton X 100 100 µg/ml cycloheximide cOmplete protease inhibitor cocktail [1 tablet per 10 ml buffer] and 200 units/ml SUPERase•In in a volume of 10 µl per oocyte/embryo by vigorous shaking and pipetting. Lysates were cleared by centrifugation at 5000 g at 4°C for 10 min. The supernatant was transfered to a new tube and mixed with the Tri reagent for RNA isolation. Fish embryos were de chorionated by incubation with 2 mg/ml pronase in E3 medium for 4 min. post removing all E3 medium Tri Reagent was added to the embryos for RNA isolation. Mouse GV oocytes were collected in 37°C MEM in the presence of milrinone to prevent maturation. Cumulus cells were removed from cumulus oocyte complexes by repeated aspiration through a glass pipette. GV oocytes were then collected in TRI reagent for RNA isolation. Mouse MII oocytes were harvested from the oviducts of hCG induced mice 16 hr denuded with 3 mg/ml hyaluronidase for 2 min washed and then collected in Tri reagent for RNA isolation. For gene specific tail seq of reporter libraries total RNA with reporter mRNA libraries was ligated to a pre adenylated 3ʹ adapter directly in most cases but for the N60 library injected into fish embryos and frog oocytes the N37 PAS N17 library injected into fish embryos and frog oocytes and the CPEmos N60 and N60LC PASmos libraries injected into frog oocytes reporter library mRNAs were enriched by anti sense oligo capture with biotinylated oligos. RNA isolated from the oocyte or embryo lysate was mixed with 8 pmol KXSH009 8 pmol KXSH010 and 2x SSC 0.3 M NaCl 30 mM sodium citrate pH 7.0 in a total of 50 µl. The RNA and oligos were annealed by incubation at 70°C for 5 min and then slowly cooling to 23°C at 0.1°C/sec. The annealed mixture was combined with 40 µl MyOne Streptavidin C1 beads Thermo Fisher 65002 and incubated for 20 min at 23°C on a thermal mixer shaking with 15 sec on and 1 min 45 sec off. The superna…   tissue:embryo|treatment:N1 GSM7716865 GSM7716865: Fish embryo mRNA 1cell PAL seq v4; Danio rerio; OTHER GSM7716865 r1 GSM7716865 1 Frog oocytes and embryos were lysed in ice cold buffer RL 20 mM HEPES pH 7.5 100 mM KCl 5 mM MgCl2 1% [v/v] Triton X 100 100 µg/ml cycloheximide cOmplete protease inhibitor cocktail [1 tablet per 10 ml buffer] and 200 units/ml SUPERase•In in a volume of 10 µl per oocyte/embryo by vigorous shaking and pipetting. Lysates were cleared by centrifugation at 5000 g at 4°C for 10 min. The supernatant was transfered to a new tube and mixed with the Tri reagent for RNA isolation. Fish embryos were de chorionated by incubation with 2 mg/ml pronase in E3 medium for 4 min. post removing all E3 medium Tri Reagent was added to the embryos for RNA isolation. Mouse GV oocytes were collected in 37°C MEM in the presence of milrinone to prevent maturation. Cumulus cells were removed from cumulus oocyte complexes by repeated aspiration through a glass pipette. GV oocytes were then collected in TRI reagent for RNA isolation. Mouse MII oocytes were harvested from the oviducts of hCG induced mice 16 hr denuded with 3 mg/ml hyaluronidase for 2 min washed and then collected in Tri reagent for RNA isolation. For gene specific tail seq of reporter libraries total RNA with reporter mRNA libraries was ligated to a pre adenylated 3ʹ adapter directly in most cases but for the N60 library injected into fish embryos and frog oocytes the N37 PAS N17 library injected into fish embryos and frog oocytes and the CPEmos N60 and N60LC PASmos libraries injected into frog oocytes reporter library mRNAs were enriched by anti sense oligo capture with biotinylated oligos. RNA isolated from the oocyte or embryo lysate was mixed with 8 pmol KXSH009 8 pmol KXSH010 and 2x SSC 0.3 M NaCl 30 mM sodium citrate pH 7.0 in a total of 50 µl. The RNA and oligos were annealed by incubation at 70°C for 5 min and then slowly cooling to 23°C at 0.1°C/sec. The annealed mixture was combined with 40 µl MyOne Streptavidin C1 beads Thermo Fisher 65002 and incubated for 20 min at 23°C on a thermal mixer shaking with 15 sec on and 1 min 45 sec off. The superna…   OTHER TRANSCRIPTOMIC other PAIRED ILLUMINA Illumina HiSeq 2500   SRP455680     Fish_embryo_mRNA_1cell_PAL_seq_v4_rep2_raw_read1.fastq.gz Fish_embryo_mRNA_1cell_PAL_seq_v4_rep2_raw_read2.fastq.gz fastq fastq 3406093776.0 11094768.0 GSM7716865 r2 0:52 1:255 A:868054279;C:890446070;G:945289336;T:695324057;N:6980034 52 255     868054279 890446070 945289336 695324057 6980034 SRX21396036 SRS18636194 SRA1694849 Whitehead Institute Whitehead Institute 2 0.00049 0.0 0.00014 0.0 0.99939 1.0 0.58974   52 255 T T mates < 9% mapping rate illumina hiseq_era unknown poly_a unknown bulk bulk bulk   United States 2023-08-17 Zygote Embryo Embryo Imprecise All anatomical structures
25295 25295 SRR25764045 SRX21486723 SRS18719024 SRP457105 PRJNA1009809 Dynamics of the zebrafish tRNAome during the maternal to zygotic transition [Ribo Seq] GSE241753 Other Time course analysis of tRNA abundance during zebrafish early embryonic development. Overall design: Wild type TLAB strain zebrafish embryos were grown in standard housing conditions.Unfertilized eggs 0 hpf were collected or embryos were staged and collected at consecutive developmental time points namelyat the 256 cell 2.5 hpf 1000 cell 3 hpf sphere 4 hpf shield 6 hpf and bud 10 hpf stages. Eggs and embryos were either flash frozen in liquid nitrogen or immediately processed. Samples were used for western blotting analysis polysome profiling ribosome profiling or mRNA and tRNA sequencing for investigating the regulation of tRNA gene expression and translational status during early zebrafish embryogenesis. parent bioproject:PRJNA1009800 pubmed:39402326   WT bud 10 hpf Ribo seq rep1 GSM7734770   source name:Gastrula|strain:TLAB strain|tissue:Gastrula|developmental stage:Bud 10 hpf|treatment:100 µg/ml CHX 100 µg/ml TIG|geo loc name:missing|collection date:missing WT bud 10 hpf Ribo seq rep1 We identified the A site location in each mapped read with Scikit ribo [Fang et al. 2018] which uses a random forest with recursive feature selection and a generalized linear model for accurate A site prediction based on matched ribosome profiling and RNA Seq datasets. Kallisto 0.44.0 with parameters b 100 single l 180 s 20 t 40 was used to quantify transcript abundances in Transcripts Per Million TPM from RNA Seq data based on the reference set of MANE annotated transcripts see Codon usage analysis for description of this annotation. To avoid memory errors due to the large size of the human genome and the presence of multiple transcript isoforms all RNAfold dependencies in Scikit ribo were omitted and the index was built separately for each chromosome. To make the hg38 GTF compatible with Scikit ribo transcript/UTR annotations were removed. For each transcript the start codon in the first exon and the stop codon in the last exon were adjusted to represent transcript start and end coordinates taking into account the gene strand. To estimate codon dwell times short 20 23 nt and long 28 33 nt ribosome footprints were analyzed separately. rRNA filtered reads were aligned to GRCz11.108 using STAR v2.6.1c [Dobin et al. 2013] the following options: outFilterMultimapNmax 1 seedSearchStartLmax 15 outSAMtype BAM SortedByCoordinate outFilterMismatchNmax 2 alignEndsType EndToEnd quantMode TranscriptomeSAM outSAMattributes NH HI AS nM NM MD We identified the A site location in each mapped read with Scikit ribo [Fang et al. 2018] which uses a random forest with recursive feature selection and a generalized linear model for accurate A site prediction based on matched ribosome profiling and RNA Seq datasets. Kallisto 0.44.0 with parameters b 100 single l 180 s 20 t 40 was used to quantify transcript abundances in Transcripts Per Million TPM from RNA Seq data based on the reference set of MANE annotated transcripts see Codon usage analysis for description of this annotation. To avoid memory err… Gastrula unperturbed growth conditions in E3 medium for zebrafish embryos. 200 whole embryos were flash frozen in liquid nitrogen and subsequently lysed in footprint lysis buffer containing 100 µg/ml CHX and 100 µg/ml TIG 0.1% NP 40 10 µg/ml aprotinin 20 µM leupeptin 2.5 µM pepstatin A 0.5 mM AEBSF and 1x Phosphatase Inhibitor Cocktail. Samples were vortexed vigorously triturated through a 26G gauge needle and spun down for 7 minutes at 16 000xg/ 4°C. Supernatant was transferred to a new tube. 20 µg RNA in 200 µl polysome lysis buffer were digested with 50 U RNase I for 45 minutes at 2 000 rpm/22°C. post incubation on ice for 5 minutes extracts were pre cleared by centrifugation for 5 minutes at 3 000 g/ 4°C. Ribosomes were pelleted through 3 ml of a sucrose cushion 1 M sucrose 20 mM Tris pH=8.0 140 mM KCl 5 mM MgCl2 1 mM DTT by spinning the layered solutions in the Type 70 Ti rotor for 120 minutes at 50 000 rpm/ 4°C. Ribosome pellets were rinsed once dissolved in 200 µl drug free polysome lysis buffer and incubated with 200 U hiPSC or 300 U NPC RNase I for 45 minutes at 2 000 rpm/22°C. Ribosome footprint libraries were prepared essentially as described McGlincy and Ingolia 2017; Wu 2019 with minor modifications. RNase I digestion was stopped by addition of 100 U Superase In and extracts were loaded on a sucrose cushion. The pellet was dissolved in 400 µl LiDS/LET lysis buffer and RNA was extracted with the acid phenol protocol. Fragments in the range of 19 to 32 nucleotides were isolated by gel size selection with T4 PNK and ligated to pre adenylated adapters containing 5 random nucleotides at their 5’ ends McGlinzy 2017 with T4 RNA Ligase 2 truncated KQ. Adapter ligated RNA was subjected to rRNA depletion using the Ribo Seq riboPOOL h/m/r depletion kit siTOOLs for CHX only samples and legacy RiboZero Gold kit Illumina for CHX+TIG samples The rRNA depleted footprints were reverse transcribed with Protoscript II and cDNA was circularized with recombinant TS2126 RNA ligase 1 commercially available as CircLigase. Libraries were constructed from circularized cDNA with KAP… Embryos were grown in standard housing conditions namely28°C at a 14/10 hour light/dark cycle. strain:TLAB strain|tissue:Gastrula|developmental stage:Bud 10 hpf|treatment:100 µg/ml CHX 100 µg/ml TIG GSM7734770 GSM7734770: WT bud 10 hpf Ribo seq rep1; Danio rerio; OTHER GSM7734770 r1 GSM7734770 1 200 whole embryos were flash frozen in liquid nitrogen and subsequently lysed in footprint lysis buffer containing 100 µg/ml CHX and 100 µg/ml TIG 0.1% NP 40 10 µg/ml aprotinin 20 µM leupeptin 2.5 µM pepstatin A 0.5 mM AEBSF and 1x Phosphatase Inhibitor Cocktail. Samples were vortexed vigorously triturated through a 26G gauge needle and spun down for 7 minutes at 16 000xg/ 4°C. Supernatant was transferred to a new tube. 20 µg RNA in 200 µl polysome lysis buffer were digested with 50 U RNase I for 45 minutes at 2 000 rpm/22°C. post incubation on ice for 5 minutes extracts were pre cleared by centrifugation for 5 minutes at 3 000 g/ 4°C. Ribosomes were pelleted through 3 ml of a sucrose cushion 1 M sucrose 20 mM Tris pH=8.0 140 mM KCl 5 mM MgCl2 1 mM DTT by spinning the layered solutions in the Type 70 Ti rotor for 120 minutes at 50 000 rpm/ 4°C. Ribosome pellets were rinsed once dissolved in 200 µl drug free polysome lysis buffer and incubated with 200 U hiPSC or 300 U NPC RNase I for 45 minutes at 2 000 rpm/22°C. Ribosome footprint libraries were prepared essentially as described McGlincy and Ingolia 2017; Wu 2019 with minor modifications. RNase I digestion was stopped by addition of 100 U Superase In and extracts were loaded on a sucrose cushion. The pellet was dissolved in 400 µl LiDS/LET lysis buffer and RNA was extracted with the acid phenol protocol. Fragments in the range of 19 to 32 nucleotides were isolated by gel size selection with T4 PNK and ligated to pre adenylated adapters containing 5 random nucleotides at their five prime ends McGlinzy 2017 with T4 RNA Ligase 2 truncated KQ. Adapter ligated RNA was subjected to rRNA depletion using the Ribo Seq riboPOOL h/m/r depletion kit siTOOLs for CHX only samples and legacy RiboZero Gold kit Illumina for CHX+TIG samples The rRNA depleted footprints were reverse transcribed with Protoscript II and cDNA was circularized with recombinant TS2126 RNA ligase 1 commercially available as CircLigase. Libraries were constructed from circularized cDNA …   OTHER TRANSCRIPTOMIC other SINGLE ILLUMINA NextSeq 500   SRP457105     WT_ribo_bud_1.fastq.gz fastq 1476985703.0 55745002.0 GSM7734770 r1 0:26.50 A:266369385;C:466461341;G:473469631;T:270671671;N:13675 26       266369385 466461341 473469631 270671671 13675 SRX21486723 SRS18719024 SRA1700409 Mechanisms of Protein Biogenesis, Max Planck Institute for Biochemistry Max Planck Institute of Biochemistry 1 0.773   0.14121   0.82242   0.78464   30   B   usable mapping rate illumina nextseq 5prime rrna_depletion ribozero bulk unknown unknown   Germany 2023-08-28 Gastrula Embryo Whole Organism All anatomical structures
25296 25296 SRR25764046 SRX21486722 SRS18719023 SRP457105 PRJNA1009809 Dynamics of the zebrafish tRNAome during the maternal to zygotic transition [Ribo Seq] GSE241753 Other Time course analysis of tRNA abundance during zebrafish early embryonic development. Overall design: Wild type TLAB strain zebrafish embryos were grown in standard housing conditions.Unfertilized eggs 0 hpf were collected or embryos were staged and collected at consecutive developmental time points namelyat the 256 cell 2.5 hpf 1000 cell 3 hpf sphere 4 hpf shield 6 hpf and bud 10 hpf stages. Eggs and embryos were either flash frozen in liquid nitrogen or immediately processed. Samples were used for western blotting analysis polysome profiling ribosome profiling or mRNA and tRNA sequencing for investigating the regulation of tRNA gene expression and translational status during early zebrafish embryogenesis. parent bioproject:PRJNA1009800 pubmed:39402326   WT sphere 4 hpf Ribo seq rep1 GSM7734769   source name:Blastula|strain:TLAB strain|tissue:Blastula|developmental stage:Sphere 4 hpf|treatment:100 µg/ml CHX 100 µg/ml TIG|geo loc name:missing|collection date:missing WT sphere 4 hpf Ribo seq rep1 We identified the A site location in each mapped read with Scikit ribo [Fang et al. 2018] which uses a random forest with recursive feature selection and a generalized linear model for accurate A site prediction based on matched ribosome profiling and RNA Seq datasets. Kallisto 0.44.0 with parameters b 100 single l 180 s 20 t 40 was used to quantify transcript abundances in Transcripts Per Million TPM from RNA Seq data based on the reference set of MANE annotated transcripts see Codon usage analysis for description of this annotation. To avoid memory errors due to the large size of the human genome and the presence of multiple transcript isoforms all RNAfold dependencies in Scikit ribo were omitted and the index was built separately for each chromosome. To make the hg38 GTF compatible with Scikit ribo transcript/UTR annotations were removed. For each transcript the start codon in the first exon and the stop codon in the last exon were adjusted to represent transcript start and end coordinates taking into account the gene strand. To estimate codon dwell times short 20 23 nt and long 28 33 nt ribosome footprints were analyzed separately. rRNA filtered reads were aligned to GRCz11.108 using STAR v2.6.1c [Dobin et al. 2013] the following options: outFilterMultimapNmax 1 seedSearchStartLmax 15 outSAMtype BAM SortedByCoordinate outFilterMismatchNmax 2 alignEndsType EndToEnd quantMode TranscriptomeSAM outSAMattributes NH HI AS nM NM MD We identified the A site location in each mapped read with Scikit ribo [Fang et al. 2018] which uses a random forest with recursive feature selection and a generalized linear model for accurate A site prediction based on matched ribosome profiling and RNA Seq datasets. Kallisto 0.44.0 with parameters b 100 single l 180 s 20 t 40 was used to quantify transcript abundances in Transcripts Per Million TPM from RNA Seq data based on the reference set of MANE annotated transcripts see Codon usage analysis for description of this annotation. To avoid memory err… Blastula unperturbed growth conditions in E3 medium for zebrafish embryos. 200 whole embryos were flash frozen in liquid nitrogen and subsequently lysed in footprint lysis buffer containing 100 µg/ml CHX and 100 µg/ml TIG 0.1% NP 40 10 µg/ml aprotinin 20 µM leupeptin 2.5 µM pepstatin A 0.5 mM AEBSF and 1x Phosphatase Inhibitor Cocktail. Samples were vortexed vigorously triturated through a 26G gauge needle and spun down for 7 minutes at 16 000xg/ 4°C. Supernatant was transferred to a new tube. 20 µg RNA in 200 µl polysome lysis buffer were digested with 50 U RNase I for 45 minutes at 2 000 rpm/22°C. post incubation on ice for 5 minutes extracts were pre cleared by centrifugation for 5 minutes at 3 000 g/ 4°C. Ribosomes were pelleted through 3 ml of a sucrose cushion 1 M sucrose 20 mM Tris pH=8.0 140 mM KCl 5 mM MgCl2 1 mM DTT by spinning the layered solutions in the Type 70 Ti rotor for 120 minutes at 50 000 rpm/ 4°C. Ribosome pellets were rinsed once dissolved in 200 µl drug free polysome lysis buffer and incubated with 200 U hiPSC or 300 U NPC RNase I for 45 minutes at 2 000 rpm/22°C. Ribosome footprint libraries were prepared essentially as described McGlincy and Ingolia 2017; Wu 2019 with minor modifications. RNase I digestion was stopped by addition of 100 U Superase In and extracts were loaded on a sucrose cushion. The pellet was dissolved in 400 µl LiDS/LET lysis buffer and RNA was extracted with the acid phenol protocol. Fragments in the range of 19 to 32 nucleotides were isolated by gel size selection with T4 PNK and ligated to pre adenylated adapters containing 5 random nucleotides at their 5’ ends McGlinzy 2017 with T4 RNA Ligase 2 truncated KQ. Adapter ligated RNA was subjected to rRNA depletion using the Ribo Seq riboPOOL h/m/r depletion kit siTOOLs for CHX only samples and legacy RiboZero Gold kit Illumina for CHX+TIG samples The rRNA depleted footprints were reverse transcribed with Protoscript II and cDNA was circularized with recombinant TS2126 RNA ligase 1 commercially available as CircLigase. Libraries were constructed from circularized cDNA with KAP… Embryos were grown in standard housing conditions namely28°C at a 14/10 hour light/dark cycle. strain:TLAB strain|tissue:Blastula|developmental stage:Sphere 4 hpf|treatment:100 µg/ml CHX 100 µg/ml TIG GSM7734769 GSM7734769: WT sphere 4 hpf Ribo seq rep1; Danio rerio; OTHER GSM7734769 r1 GSM7734769 1 200 whole embryos were flash frozen in liquid nitrogen and subsequently lysed in footprint lysis buffer containing 100 µg/ml CHX and 100 µg/ml TIG 0.1% NP 40 10 µg/ml aprotinin 20 µM leupeptin 2.5 µM pepstatin A 0.5 mM AEBSF and 1x Phosphatase Inhibitor Cocktail. Samples were vortexed vigorously triturated through a 26G gauge needle and spun down for 7 minutes at 16 000xg/ 4°C. Supernatant was transferred to a new tube. 20 µg RNA in 200 µl polysome lysis buffer were digested with 50 U RNase I for 45 minutes at 2 000 rpm/22°C. post incubation on ice for 5 minutes extracts were pre cleared by centrifugation for 5 minutes at 3 000 g/ 4°C. Ribosomes were pelleted through 3 ml of a sucrose cushion 1 M sucrose 20 mM Tris pH=8.0 140 mM KCl 5 mM MgCl2 1 mM DTT by spinning the layered solutions in the Type 70 Ti rotor for 120 minutes at 50 000 rpm/ 4°C. Ribosome pellets were rinsed once dissolved in 200 µl drug free polysome lysis buffer and incubated with 200 U hiPSC or 300 U NPC RNase I for 45 minutes at 2 000 rpm/22°C. Ribosome footprint libraries were prepared essentially as described McGlincy and Ingolia 2017; Wu 2019 with minor modifications. RNase I digestion was stopped by addition of 100 U Superase In and extracts were loaded on a sucrose cushion. The pellet was dissolved in 400 µl LiDS/LET lysis buffer and RNA was extracted with the acid phenol protocol. Fragments in the range of 19 to 32 nucleotides were isolated by gel size selection with T4 PNK and ligated to pre adenylated adapters containing 5 random nucleotides at their five prime ends McGlinzy 2017 with T4 RNA Ligase 2 truncated KQ. Adapter ligated RNA was subjected to rRNA depletion using the Ribo Seq riboPOOL h/m/r depletion kit siTOOLs for CHX only samples and legacy RiboZero Gold kit Illumina for CHX+TIG samples The rRNA depleted footprints were reverse transcribed with Protoscript II and cDNA was circularized with recombinant TS2126 RNA ligase 1 commercially available as CircLigase. Libraries were constructed from circularized cDNA …   OTHER TRANSCRIPTOMIC other SINGLE ILLUMINA NextSeq 500   SRP457105     WT_ribo_sphere_1.fastq.gz fastq 1336027898.0 47907512.0 GSM7734769 r1 0:27.89 A:229038284;C:439455963;G:429428292;T:238088512;N:16847 27       229038284 439455963 429428292 238088512 16847 SRX21486722 SRS18719023 SRA1700409 Mechanisms of Protein Biogenesis, Max Planck Institute for Biochemistry Max Planck Institute of Biochemistry 1 0.85867   0.20418   0.8776   0.79481   24   B   usable mapping rate illumina nextseq 5prime rrna_depletion ribozero bulk unknown unknown   Germany 2023-08-28 Blastula Embryo Whole Organism All anatomical structures
28725 28725 SRR26623262 SRX22323921 SRS19374450 SRP469552 PRJNA1034159 Dissecting the spatiotemporal diversity of adult neural stem cells GSE246714 Other Adult stem cells are important for tissue turnover and regeneration. However in most adult systems it remains elusive how stem cells assume different functional states and support spatially patterned tissue architecture. Here we dissected the diversity of neural stem cells in the adult zebrafish brain an organ that is characterized by pronounced zonation and high regenerative capacity. We combined single cell transcriptomics of dissected brain regions with massively parallel lineage tracing and in vivo RNA metabolic labeling to analyze regulation of neural stem cells in space and time. We detected a large diversity of neural stem cells with some subtypes being restricted to a single brain region while others were found globally across the brain. Global stem cell states are linked to neurogenic differentiation with different states being involved in proliferative and non proliferative differentiation. Our work reveals principles of adult stem cell organization and establishes a resource for functional manipulation of neural stem cell subtypes. Overall design: Single cell RNA seq of adult zebrafish brain performed using 10x Genomics Gene Expression protocols to explore transcriptomic diversity of adult cell types. The samples were prepared either from whole brain FACS sorted cells GFP positive cells from gfap:GFP line or a dissected single brain region as indicated in the sample title. CRISPR based lineage tracing was performed for a subset of samples to dissect developmental lineage relationships between cells. ScSLAM seq was performed in combination with Notch pathway inhibition to assess response of stem cells to perturbation.   pubmed:38365956   Lineage tracing rep1 cirbpb scars GSM7875190   source name:adult brain|tissue:adult brain|cell type:mixed tissue dissociation|genotype:Tg[ubi:zebrabow M]|geo loc name:missing|collection date:missing Lineage tracing rep1 cirbpb scars The transcriptome libraries were demultiplexed and mapped with CellRanger 6.1.1. In order to reduce batch effect the gene expression matrices were processed with the SoupX tool Young & Behjati 2020 for removing ambient RNA contamination. A subset of libraries was also mapped with Velocyto v0.17.17 La Manno et al 2018 to create count matrices for unspliced and spliced molecules. The targeted lineage tracing libraries were aligned using bwa mem3 v.0.7.12 to individual references of endogenous genes used in the lineage tracing experiment actb1 actb2 cfl1 cirbpb rpl39 and ube2e1. The scars were filtered with a custom pipeline to eliminate sequences that originate from PCR or sequencing errors. For custom code and further downstream analysis see: https://github.com/nimitic/radial glia. Assembly: GRCz11 Supplementary files format and content: Tab separated barcode files and matrix files along with the tab separated gene file supplementary file are the output of CellRanger mapping further processed by the SoupX package for ambient RNA removal. Supplementary files format and content: Loom files are the output of velocyto mapping distinguishing spliced and unspliced mRNAs. Supplementary files format and content: filtered scars.csv files contain the output of a custom scar filtering pipeline. adult brain   Adult zebrafish brain tissue was extracted optionally dissected to select a specific region of interest telencephalon diencephalon mesencephalon or rhombencephalon and then dissociated with a trypsin protocol. Briefly the samples were incubated in 750 µl of HBSS glucose solution and dissocated with 15 µl of 0.05% trypsin EDTA Gibco for 30 minutes at 37°C with intermittent mixing. The disociation was stopped by addition of 750 µl of a BSA EBSS HEPES solution. Further sample handling steps were carried out on ice and for centrifugation at 4°C. The sample was filtered through a 100 µm filter washed with cold HBSS and resuspended in 100 200 µl HBSS with 0.05% BSA then filtered through a 35 µm filter and loaded on the 10x Chromium Controller. For the scSLAM seq samples a papain dissociation protocol Worthington kit according to manufecturer's instructions was used followed by an adapted scSLAM seq protocol for labeling nascent transcripts see sample specific entry. The standard transcriptome libraries were constructed according to the manufacturer's protocol 10X Genomics. For targeted libraries of scars used for lineage tracing the cDNA obtained during the 10x protocol was used to set up individual PCR reactions for each target specifically amplifying the sgRNA target site. The libraries for targeted PCR were constructed with primers compatible with the 10X Genomics kit so they could be sequenced on the same platform.   tissue:adult brain|cell type:mixed tissue dissociation|genotype:Tg[ubi:zebrabow M] GSM7875190 GSM7875190: Lineage tracing rep1 cirbpb scars; Danio rerio; OTHER GSM7875190 r1 GSM7875190 1 Adult zebrafish brain tissue was extracted optionally dissected to select a specific region of interest telencephalon diencephalon mesencephalon or rhombencephalon and then dissociated with a trypsin protocol. Briefly the samples were incubated in 750 µl of HBSS glucose solution and dissocated with 15 µl of 0.05% trypsin EDTA Gibco for 30 minutes at 37°C with intermittent mixing. The disociation was stopped by addition of 750 µl of a BSA EBSS HEPES solution. Further sample handling steps were carried out on ice and for centrifugation at 4°C. The sample was filtered through a 100 µm filter washed with cold HBSS and resuspended in 100 200 µl HBSS with 0.05% BSA then filtered through a 35 µm filter and loaded on the 10x Chromium Controller. For the scSLAM seq samples a papain dissociation protocol Worthington kit according to manufecturer's instructions was used followed by an adapted scSLAM seq protocol for labeling nascent transcripts see sample specific entry. The standard transcriptome libraries were constructed according to the manufacturer's protocol 10X Genomics. For targeted libraries of scars used for lineage tracing the cDNA obtained during the 10x protocol was used to set up individual PCR reactions for each target specifically amplifying the sgRNA target site. The libraries for targeted PCR were constructed with primers compatible with the 10X Genomics kit so they could be sequenced on the same platform.   OTHER TRANSCRIPTOMIC other PAIRED ILLUMINA NextSeq 500   SRP469552   loader:fastq load.py lin1_cirbpb_scar_R1.fastq.gz lin1_cirbpb_scar_R2.fastq.gz fastq fastq 157576336.0 949552.0 GSM7875190 r1                       SRX22323921 SRS19374450 SRA1743007 Junker Lab, Berlin Institute for Medical Systems Biology, Max-Delbrück-Center for Molecular Medicine Junker Lab, Berlin Institute for Medical Systems Biology, Max-Delbrück-Center for Molecular Medicine 2 0.00141 0.78069 0.00053 0.00877 0.99857 0.97822 0.33536 0.05199 28 120 T B sc-like readlen illumina nextseq unknown other unknown sc single_cell_droplet 10x   Germany 2023-10-31 Adult Adult Brain Nervous System
28726 28726 SRR26623263 SRX22323920 SRS19374449 SRP469552 PRJNA1034159 Dissecting the spatiotemporal diversity of adult neural stem cells GSE246714 Other Adult stem cells are important for tissue turnover and regeneration. However in most adult systems it remains elusive how stem cells assume different functional states and support spatially patterned tissue architecture. Here we dissected the diversity of neural stem cells in the adult zebrafish brain an organ that is characterized by pronounced zonation and high regenerative capacity. We combined single cell transcriptomics of dissected brain regions with massively parallel lineage tracing and in vivo RNA metabolic labeling to analyze regulation of neural stem cells in space and time. We detected a large diversity of neural stem cells with some subtypes being restricted to a single brain region while others were found globally across the brain. Global stem cell states are linked to neurogenic differentiation with different states being involved in proliferative and non proliferative differentiation. Our work reveals principles of adult stem cell organization and establishes a resource for functional manipulation of neural stem cell subtypes. Overall design: Single cell RNA seq of adult zebrafish brain performed using 10x Genomics Gene Expression protocols to explore transcriptomic diversity of adult cell types. The samples were prepared either from whole brain FACS sorted cells GFP positive cells from gfap:GFP line or a dissected single brain region as indicated in the sample title. CRISPR based lineage tracing was performed for a subset of samples to dissect developmental lineage relationships between cells. ScSLAM seq was performed in combination with Notch pathway inhibition to assess response of stem cells to perturbation.   pubmed:38365956   Lineage tracing rep1 cfl1 scars GSM7875189   source name:adult brain|tissue:adult brain|cell type:mixed tissue dissociation|genotype:Tg[ubi:zebrabow M]|geo loc name:missing|collection date:missing Lineage tracing rep1 cfl1 scars The transcriptome libraries were demultiplexed and mapped with CellRanger 6.1.1. In order to reduce batch effect the gene expression matrices were processed with the SoupX tool Young & Behjati 2020 for removing ambient RNA contamination. A subset of libraries was also mapped with Velocyto v0.17.17 La Manno et al 2018 to create count matrices for unspliced and spliced molecules. The targeted lineage tracing libraries were aligned using bwa mem3 v.0.7.12 to individual references of endogenous genes used in the lineage tracing experiment actb1 actb2 cfl1 cirbpb rpl39 and ube2e1. The scars were filtered with a custom pipeline to eliminate sequences that originate from PCR or sequencing errors. For custom code and further downstream analysis see: https://github.com/nimitic/radial glia. Assembly: GRCz11 Supplementary files format and content: Tab separated barcode files and matrix files along with the tab separated gene file supplementary file are the output of CellRanger mapping further processed by the SoupX package for ambient RNA removal. Supplementary files format and content: Loom files are the output of velocyto mapping distinguishing spliced and unspliced mRNAs. Supplementary files format and content: filtered scars.csv files contain the output of a custom scar filtering pipeline. adult brain   Adult zebrafish brain tissue was extracted optionally dissected to select a specific region of interest telencephalon diencephalon mesencephalon or rhombencephalon and then dissociated with a trypsin protocol. Briefly the samples were incubated in 750 µl of HBSS glucose solution and dissocated with 15 µl of 0.05% trypsin EDTA Gibco for 30 minutes at 37°C with intermittent mixing. The disociation was stopped by addition of 750 µl of a BSA EBSS HEPES solution. Further sample handling steps were carried out on ice and for centrifugation at 4°C. The sample was filtered through a 100 µm filter washed with cold HBSS and resuspended in 100 200 µl HBSS with 0.05% BSA then filtered through a 35 µm filter and loaded on the 10x Chromium Controller. For the scSLAM seq samples a papain dissociation protocol Worthington kit according to manufecturer's instructions was used followed by an adapted scSLAM seq protocol for labeling nascent transcripts see sample specific entry. The standard transcriptome libraries were constructed according to the manufacturer's protocol 10X Genomics. For targeted libraries of scars used for lineage tracing the cDNA obtained during the 10x protocol was used to set up individual PCR reactions for each target specifically amplifying the sgRNA target site. The libraries for targeted PCR were constructed with primers compatible with the 10X Genomics kit so they could be sequenced on the same platform.   tissue:adult brain|cell type:mixed tissue dissociation|genotype:Tg[ubi:zebrabow M] GSM7875189 GSM7875189: Lineage tracing rep1 cfl1 scars; Danio rerio; OTHER GSM7875189 r1 GSM7875189 1 Adult zebrafish brain tissue was extracted optionally dissected to select a specific region of interest telencephalon diencephalon mesencephalon or rhombencephalon and then dissociated with a trypsin protocol. Briefly the samples were incubated in 750 µl of HBSS glucose solution and dissocated with 15 µl of 0.05% trypsin EDTA Gibco for 30 minutes at 37°C with intermittent mixing. The disociation was stopped by addition of 750 µl of a BSA EBSS HEPES solution. Further sample handling steps were carried out on ice and for centrifugation at 4°C. The sample was filtered through a 100 µm filter washed with cold HBSS and resuspended in 100 200 µl HBSS with 0.05% BSA then filtered through a 35 µm filter and loaded on the 10x Chromium Controller. For the scSLAM seq samples a papain dissociation protocol Worthington kit according to manufecturer's instructions was used followed by an adapted scSLAM seq protocol for labeling nascent transcripts see sample specific entry. The standard transcriptome libraries were constructed according to the manufacturer's protocol 10X Genomics. For targeted libraries of scars used for lineage tracing the cDNA obtained during the 10x protocol was used to set up individual PCR reactions for each target specifically amplifying the sgRNA target site. The libraries for targeted PCR were constructed with primers compatible with the 10X Genomics kit so they could be sequenced on the same platform.   OTHER TRANSCRIPTOMIC other PAIRED ILLUMINA NextSeq 500   SRP469552   loader:fastq load.py lin1_cfl1_scar_R2.fastq.gz lin1_cfl1_scar_R1.fastq.gz fastq fastq 381453696.0 2184402.0 GSM7875189 r1                       SRX22323920 SRS19374449 SRA1743007 Junker Lab, Berlin Institute for Medical Systems Biology, Max-Delbrück-Center for Molecular Medicine Junker Lab, Berlin Institute for Medical Systems Biology, Max-Delbrück-Center for Molecular Medicine 2 0.0002 0.91626 0.00014 0.00132 0.99987 0.99726 0.16666 0.56363 28 120 T B sc-like readlen illumina nextseq unknown other unknown sc single_cell_droplet 10x   Germany 2023-10-31 Adult Adult Brain Nervous System
28727 28727 SRR26623264 SRX22323919 SRS19374448 SRP469552 PRJNA1034159 Dissecting the spatiotemporal diversity of adult neural stem cells GSE246714 Other Adult stem cells are important for tissue turnover and regeneration. However in most adult systems it remains elusive how stem cells assume different functional states and support spatially patterned tissue architecture. Here we dissected the diversity of neural stem cells in the adult zebrafish brain an organ that is characterized by pronounced zonation and high regenerative capacity. We combined single cell transcriptomics of dissected brain regions with massively parallel lineage tracing and in vivo RNA metabolic labeling to analyze regulation of neural stem cells in space and time. We detected a large diversity of neural stem cells with some subtypes being restricted to a single brain region while others were found globally across the brain. Global stem cell states are linked to neurogenic differentiation with different states being involved in proliferative and non proliferative differentiation. Our work reveals principles of adult stem cell organization and establishes a resource for functional manipulation of neural stem cell subtypes. Overall design: Single cell RNA seq of adult zebrafish brain performed using 10x Genomics Gene Expression protocols to explore transcriptomic diversity of adult cell types. The samples were prepared either from whole brain FACS sorted cells GFP positive cells from gfap:GFP line or a dissected single brain region as indicated in the sample title. CRISPR based lineage tracing was performed for a subset of samples to dissect developmental lineage relationships between cells. ScSLAM seq was performed in combination with Notch pathway inhibition to assess response of stem cells to perturbation.   pubmed:38365956   Lineage tracing rep1 actb2 scars GSM7875188   source name:adult brain|tissue:adult brain|cell type:mixed tissue dissociation|genotype:Tg[ubi:zebrabow M]|geo loc name:missing|collection date:missing Lineage tracing rep1 actb2 scars The transcriptome libraries were demultiplexed and mapped with CellRanger 6.1.1. In order to reduce batch effect the gene expression matrices were processed with the SoupX tool Young & Behjati 2020 for removing ambient RNA contamination. A subset of libraries was also mapped with Velocyto v0.17.17 La Manno et al 2018 to create count matrices for unspliced and spliced molecules. The targeted lineage tracing libraries were aligned using bwa mem3 v.0.7.12 to individual references of endogenous genes used in the lineage tracing experiment actb1 actb2 cfl1 cirbpb rpl39 and ube2e1. The scars were filtered with a custom pipeline to eliminate sequences that originate from PCR or sequencing errors. For custom code and further downstream analysis see: https://github.com/nimitic/radial glia. Assembly: GRCz11 Supplementary files format and content: Tab separated barcode files and matrix files along with the tab separated gene file supplementary file are the output of CellRanger mapping further processed by the SoupX package for ambient RNA removal. Supplementary files format and content: Loom files are the output of velocyto mapping distinguishing spliced and unspliced mRNAs. Supplementary files format and content: filtered scars.csv files contain the output of a custom scar filtering pipeline. adult brain   Adult zebrafish brain tissue was extracted optionally dissected to select a specific region of interest telencephalon diencephalon mesencephalon or rhombencephalon and then dissociated with a trypsin protocol. Briefly the samples were incubated in 750 µl of HBSS glucose solution and dissocated with 15 µl of 0.05% trypsin EDTA Gibco for 30 minutes at 37°C with intermittent mixing. The disociation was stopped by addition of 750 µl of a BSA EBSS HEPES solution. Further sample handling steps were carried out on ice and for centrifugation at 4°C. The sample was filtered through a 100 µm filter washed with cold HBSS and resuspended in 100 200 µl HBSS with 0.05% BSA then filtered through a 35 µm filter and loaded on the 10x Chromium Controller. For the scSLAM seq samples a papain dissociation protocol Worthington kit according to manufecturer's instructions was used followed by an adapted scSLAM seq protocol for labeling nascent transcripts see sample specific entry. The standard transcriptome libraries were constructed according to the manufacturer's protocol 10X Genomics. For targeted libraries of scars used for lineage tracing the cDNA obtained during the 10x protocol was used to set up individual PCR reactions for each target specifically amplifying the sgRNA target site. The libraries for targeted PCR were constructed with primers compatible with the 10X Genomics kit so they could be sequenced on the same platform.   tissue:adult brain|cell type:mixed tissue dissociation|genotype:Tg[ubi:zebrabow M] GSM7875188 GSM7875188: Lineage tracing rep1 actb2 scars; Danio rerio; OTHER GSM7875188 r1 GSM7875188 1 Adult zebrafish brain tissue was extracted optionally dissected to select a specific region of interest telencephalon diencephalon mesencephalon or rhombencephalon and then dissociated with a trypsin protocol. Briefly the samples were incubated in 750 µl of HBSS glucose solution and dissocated with 15 µl of 0.05% trypsin EDTA Gibco for 30 minutes at 37°C with intermittent mixing. The disociation was stopped by addition of 750 µl of a BSA EBSS HEPES solution. Further sample handling steps were carried out on ice and for centrifugation at 4°C. The sample was filtered through a 100 µm filter washed with cold HBSS and resuspended in 100 200 µl HBSS with 0.05% BSA then filtered through a 35 µm filter and loaded on the 10x Chromium Controller. For the scSLAM seq samples a papain dissociation protocol Worthington kit according to manufecturer's instructions was used followed by an adapted scSLAM seq protocol for labeling nascent transcripts see sample specific entry. The standard transcriptome libraries were constructed according to the manufacturer's protocol 10X Genomics. For targeted libraries of scars used for lineage tracing the cDNA obtained during the 10x protocol was used to set up individual PCR reactions for each target specifically amplifying the sgRNA target site. The libraries for targeted PCR were constructed with primers compatible with the 10X Genomics kit so they could be sequenced on the same platform.   OTHER TRANSCRIPTOMIC other PAIRED ILLUMINA NextSeq 500   SRP469552   loader:fastq load.py lin1_actb2_scar_R1.fastq.gz lin1_actb2_scar_R2.fastq.gz fastq fastq 491682118.0 2851156.0 GSM7875188 r1                       SRX22323919 SRS19374448 SRA1743007 Junker Lab, Berlin Institute for Medical Systems Biology, Max-Delbrück-Center for Molecular Medicine Junker Lab, Berlin Institute for Medical Systems Biology, Max-Delbrück-Center for Molecular Medicine 2 0.00051 0.41901 0.00032 0.0029 0.99945 0.99182 0.57575 0.007 28 120 T B sc-like readlen illumina nextseq unknown other unknown sc single_cell_droplet 10x   Germany 2023-10-31 Adult Adult Brain Nervous System
28728 28728 SRR26623265 SRX22323918 SRS19374446 SRP469552 PRJNA1034159 Dissecting the spatiotemporal diversity of adult neural stem cells GSE246714 Other Adult stem cells are important for tissue turnover and regeneration. However in most adult systems it remains elusive how stem cells assume different functional states and support spatially patterned tissue architecture. Here we dissected the diversity of neural stem cells in the adult zebrafish brain an organ that is characterized by pronounced zonation and high regenerative capacity. We combined single cell transcriptomics of dissected brain regions with massively parallel lineage tracing and in vivo RNA metabolic labeling to analyze regulation of neural stem cells in space and time. We detected a large diversity of neural stem cells with some subtypes being restricted to a single brain region while others were found globally across the brain. Global stem cell states are linked to neurogenic differentiation with different states being involved in proliferative and non proliferative differentiation. Our work reveals principles of adult stem cell organization and establishes a resource for functional manipulation of neural stem cell subtypes. Overall design: Single cell RNA seq of adult zebrafish brain performed using 10x Genomics Gene Expression protocols to explore transcriptomic diversity of adult cell types. The samples were prepared either from whole brain FACS sorted cells GFP positive cells from gfap:GFP line or a dissected single brain region as indicated in the sample title. CRISPR based lineage tracing was performed for a subset of samples to dissect developmental lineage relationships between cells. ScSLAM seq was performed in combination with Notch pathway inhibition to assess response of stem cells to perturbation.   pubmed:38365956   Lineage tracing rep1 actb1 scars GSM7875187   source name:adult brain|tissue:adult brain|cell type:mixed tissue dissociation|genotype:Tg[ubi:zebrabow M]|geo loc name:missing|collection date:missing Lineage tracing rep1 actb1 scars The transcriptome libraries were demultiplexed and mapped with CellRanger 6.1.1. In order to reduce batch effect the gene expression matrices were processed with the SoupX tool Young & Behjati 2020 for removing ambient RNA contamination. A subset of libraries was also mapped with Velocyto v0.17.17 La Manno et al 2018 to create count matrices for unspliced and spliced molecules. The targeted lineage tracing libraries were aligned using bwa mem3 v.0.7.12 to individual references of endogenous genes used in the lineage tracing experiment actb1 actb2 cfl1 cirbpb rpl39 and ube2e1. The scars were filtered with a custom pipeline to eliminate sequences that originate from PCR or sequencing errors. For custom code and further downstream analysis see: https://github.com/nimitic/radial glia. Assembly: GRCz11 Supplementary files format and content: Tab separated barcode files and matrix files along with the tab separated gene file supplementary file are the output of CellRanger mapping further processed by the SoupX package for ambient RNA removal. Supplementary files format and content: Loom files are the output of velocyto mapping distinguishing spliced and unspliced mRNAs. Supplementary files format and content: filtered scars.csv files contain the output of a custom scar filtering pipeline. adult brain   Adult zebrafish brain tissue was extracted optionally dissected to select a specific region of interest telencephalon diencephalon mesencephalon or rhombencephalon and then dissociated with a trypsin protocol. Briefly the samples were incubated in 750 µl of HBSS glucose solution and dissocated with 15 µl of 0.05% trypsin EDTA Gibco for 30 minutes at 37°C with intermittent mixing. The disociation was stopped by addition of 750 µl of a BSA EBSS HEPES solution. Further sample handling steps were carried out on ice and for centrifugation at 4°C. The sample was filtered through a 100 µm filter washed with cold HBSS and resuspended in 100 200 µl HBSS with 0.05% BSA then filtered through a 35 µm filter and loaded on the 10x Chromium Controller. For the scSLAM seq samples a papain dissociation protocol Worthington kit according to manufecturer's instructions was used followed by an adapted scSLAM seq protocol for labeling nascent transcripts see sample specific entry. The standard transcriptome libraries were constructed according to the manufacturer's protocol 10X Genomics. For targeted libraries of scars used for lineage tracing the cDNA obtained during the 10x protocol was used to set up individual PCR reactions for each target specifically amplifying the sgRNA target site. The libraries for targeted PCR were constructed with primers compatible with the 10X Genomics kit so they could be sequenced on the same platform.   tissue:adult brain|cell type:mixed tissue dissociation|genotype:Tg[ubi:zebrabow M] GSM7875187 GSM7875187: Lineage tracing rep1 actb1 scars; Danio rerio; OTHER GSM7875187 r1 GSM7875187 1 Adult zebrafish brain tissue was extracted optionally dissected to select a specific region of interest telencephalon diencephalon mesencephalon or rhombencephalon and then dissociated with a trypsin protocol. Briefly the samples were incubated in 750 µl of HBSS glucose solution and dissocated with 15 µl of 0.05% trypsin EDTA Gibco for 30 minutes at 37°C with intermittent mixing. The disociation was stopped by addition of 750 µl of a BSA EBSS HEPES solution. Further sample handling steps were carried out on ice and for centrifugation at 4°C. The sample was filtered through a 100 µm filter washed with cold HBSS and resuspended in 100 200 µl HBSS with 0.05% BSA then filtered through a 35 µm filter and loaded on the 10x Chromium Controller. For the scSLAM seq samples a papain dissociation protocol Worthington kit according to manufecturer's instructions was used followed by an adapted scSLAM seq protocol for labeling nascent transcripts see sample specific entry. The standard transcriptome libraries were constructed according to the manufacturer's protocol 10X Genomics. For targeted libraries of scars used for lineage tracing the cDNA obtained during the 10x protocol was used to set up individual PCR reactions for each target specifically amplifying the sgRNA target site. The libraries for targeted PCR were constructed with primers compatible with the 10X Genomics kit so they could be sequenced on the same platform.   OTHER TRANSCRIPTOMIC other PAIRED ILLUMINA NextSeq 500   SRP469552   loader:fastq load.py lin1_actb1_scar_R1.fastq.gz lin1_actb1_scar_R2.fastq.gz fastq fastq 390405832.0 2283319.0 GSM7875187 r1                       SRX22323918 SRS19374446 SRA1743007 Junker Lab, Berlin Institute for Medical Systems Biology, Max-Delbrück-Center for Molecular Medicine Junker Lab, Berlin Institute for Medical Systems Biology, Max-Delbrück-Center for Molecular Medicine 2 0.00038 0.73548 0.00017 0.00057 0.99963 0.99571 0.29729 0.00243 28 120 T B sc-like readlen illumina nextseq unknown other unknown sc single_cell_droplet 10x   Germany 2023-10-31 Adult Adult Brain Nervous System
28729 28729 SRR26623266 SRX22323917 SRS19374447 SRP469552 PRJNA1034159 Dissecting the spatiotemporal diversity of adult neural stem cells GSE246714 Other Adult stem cells are important for tissue turnover and regeneration. However in most adult systems it remains elusive how stem cells assume different functional states and support spatially patterned tissue architecture. Here we dissected the diversity of neural stem cells in the adult zebrafish brain an organ that is characterized by pronounced zonation and high regenerative capacity. We combined single cell transcriptomics of dissected brain regions with massively parallel lineage tracing and in vivo RNA metabolic labeling to analyze regulation of neural stem cells in space and time. We detected a large diversity of neural stem cells with some subtypes being restricted to a single brain region while others were found globally across the brain. Global stem cell states are linked to neurogenic differentiation with different states being involved in proliferative and non proliferative differentiation. Our work reveals principles of adult stem cell organization and establishes a resource for functional manipulation of neural stem cell subtypes. Overall design: Single cell RNA seq of adult zebrafish brain performed using 10x Genomics Gene Expression protocols to explore transcriptomic diversity of adult cell types. The samples were prepared either from whole brain FACS sorted cells GFP positive cells from gfap:GFP line or a dissected single brain region as indicated in the sample title. CRISPR based lineage tracing was performed for a subset of samples to dissect developmental lineage relationships between cells. ScSLAM seq was performed in combination with Notch pathway inhibition to assess response of stem cells to perturbation.   pubmed:38365956   Brain 23 telencephalon Notch inhibition scSLAMseq GSM7875186   source name:adult brain|tissue:adult brain|tissue region:telencephalon|cell type:mixed tissue dissociation|genotype:wildtype|treatment:Notch inhibition DAPT|geo loc name:missing|collection date:missing Brain 23 telencephalon Notch inhibition scSLAMseq The transcriptome libraries were demultiplexed and mapped with CellRanger 6.1.1. In order to reduce batch effect the gene expression matrices were processed with the SoupX tool Young & Behjati 2020 for removing ambient RNA contamination. A subset of libraries was also mapped with Velocyto v0.17.17 La Manno et al 2018 to create count matrices for unspliced and spliced molecules. The targeted lineage tracing libraries were aligned using bwa mem3 v.0.7.12 to individual references of endogenous genes used in the lineage tracing experiment actb1 actb2 cfl1 cirbpb rpl39 and ube2e1. The scars were filtered with a custom pipeline to eliminate sequences that originate from PCR or sequencing errors. For custom code and further downstream analysis see: https://github.com/nimitic/radial glia. Assembly: GRCz11 Supplementary files format and content: Tab separated barcode files and matrix files along with the tab separated gene file supplementary file are the output of CellRanger mapping further processed by the SoupX package for ambient RNA removal. Supplementary files format and content: Loom files are the output of velocyto mapping distinguishing spliced and unspliced mRNAs. Supplementary files format and content: filtered scars.csv files contain the output of a custom scar filtering pipeline. Library strategy: scSLAM seq adult brain Notch inhibiton: zebrafish were incubated in a water bath containing system water with 50 µM DAPT Gamma Secretase Inhibitor Sigma Aldrich for 48 hours. The samples were prepared according to a scSLAM seq protocol adapted from Neuschulz et al 2023. Briefly: in order to label nascent transcripts 200 mM 4sU was delivered to fish brains by intraventricular injection 6 hours prior to sample collection. The brains were then collected and a single cell suspension was prepared by papain dissociation. The resulting cell suspension was fixed in 80% methanol and a conversion of 4sU using iodoactamide adding 111 µl 100 mM IAA to 800 µl fixed sample was done overnight. The following day the reaction was quanched with a quenching buffer containing 100 mM DTT post which the sample was washed with a wash buffer and filtered though a 35 µm filter. The sample was then loaded on a 10X Chromium Controller and processed according to the standard scRNA seq protocol. Adult zebrafish brain tissue was extracted optionally dissected to select a specific region of interest telencephalon diencephalon mesencephalon or rhombencephalon and then dissociated with a trypsin protocol. Briefly the samples were incubated in 750 µl of HBSS glucose solution and dissocated with 15 µl of 0.05% trypsin EDTA Gibco for 30 minutes at 37°C with intermittent mixing. The disociation was stopped by addition of 750 µl of a BSA EBSS HEPES solution. Further sample handling steps were carried out on ice and for centrifugation at 4°C. The sample was filtered through a 100 µm filter washed with cold HBSS and resuspended in 100 200 µl HBSS with 0.05% BSA then filtered through a 35 µm filter and loaded on the 10x Chromium Controller. For the scSLAM seq samples a papain dissociation protocol Worthington kit according to manufecturer's instructions was used followed by an adapted scSLAM seq protocol for labeling nascent transcripts see sample specific entry. The standard transcriptome libraries were constructed according to the manufacturer's protocol 10X Genomics. For targeted libraries of scars used for lineage tracing the cDNA obtained during the 10x protocol was used to set up individual PCR reactions …   tissue:adult brain|tissue region:telencephalon|cell type:mixed tissue dissociation|genotype:wildtype|treatment:Notch inhibition DAPT GSM7875186 GSM7875186: Brain 23 telencephalon Notch inhibition scSLAMseq; Danio rerio; OTHER GSM7875186 r1 GSM7875186 1 The samples were prepared according to a scSLAM seq protocol adapted from Neuschulz et al 2023. Briefly: in order to label nascent transcripts 200 mM 4sU was delivered to fish brains by intraventricular injection 6 hours prior to sample collection. The brains were then collected and a single cell suspension was prepared by papain dissociation. The resulting cell suspension was fixed in 80% methanol and a conversion of 4sU using iodoactamide adding 111 µl 100 mM IAA to 800 µl fixed sample was done overnight. The following day the reaction was quanched with a quenching buffer containing 100 mM DTT post which the sample was washed with a wash buffer and filtered though a 35 µm filter. The sample was then loaded on a 10X Chromium Controller and processed according to the standard scRNA seq protocol. Adult zebrafish brain tissue was extracted optionally dissected to select a specific region of interest telencephalon diencephalon mesencephalon or rhombencephalon and then dissociated with a trypsin protocol. Briefly the samples were incubated in 750 µl of HBSS glucose solution and dissocated with 15 µl of 0.05% trypsin EDTA Gibco for 30 minutes at 37°C with intermittent mixing. The disociation was stopped by addition of 750 µl of a BSA EBSS HEPES solution. Further sample handling steps were carried out on ice and for centrifugation at 4°C. The sample was filtered through a 100 µm filter washed with cold HBSS and resuspended in 100 200 µl HBSS with 0.05% BSA then filtered through a 35 µm filter and loaded on the 10x Chromium Controller. For the scSLAM seq samples a papain dissociation protocol Worthington kit according to manufecturer's instructions was used followed by an adapted scSLAM seq protocol for labeling nascent transcripts see sample specific entry. The standard transcriptome libraries were constructed according to the manufacturer's protocol 10X Genomics. For targeted libraries of scars used for lineage tracing the cDNA obtained during the 10x protocol was used to set up individual PCR reactions …   OTHER TRANSCRIPTOMIC other PAIRED ILLUMINA Illumina NovaSeq 6000   SRP469552   loader:fastq load.py b23_ni_tre_R1.fastq.gz b23_ni_tre_R2.fastq.gz fastq fastq 62392458570.0 271271559.0 GSM7875186 r1 0:28 1:202 A:18682905645;C:13783082970;G:14751373116;T:15161083446;N:14013393 28 202     18682905645 13783082970 14751373116 15161083446 14013393 SRX22323917 SRS19374447 SRA1743007 Junker Lab, Berlin Institute for Medical Systems Biology, Max-Delbrück-Center for Molecular Medicine Junker Lab, Berlin Institute for Medical Systems Biology, Max-Delbrück-Center for Molecular Medicine 2 0.01404 0.82351 0.00466 0.1026 0.99129 0.86774 0.36033 0.67345 28 202 T B sc-like readlen illumina novaseq_era unknown other unknown sc single_cell_droplet 10x   Germany 2023-10-31 Adult Adult Brain Nervous System
28730 28730 SRR26623267 SRX22323916 SRS19374445 SRP469552 PRJNA1034159 Dissecting the spatiotemporal diversity of adult neural stem cells GSE246714 Other Adult stem cells are important for tissue turnover and regeneration. However in most adult systems it remains elusive how stem cells assume different functional states and support spatially patterned tissue architecture. Here we dissected the diversity of neural stem cells in the adult zebrafish brain an organ that is characterized by pronounced zonation and high regenerative capacity. We combined single cell transcriptomics of dissected brain regions with massively parallel lineage tracing and in vivo RNA metabolic labeling to analyze regulation of neural stem cells in space and time. We detected a large diversity of neural stem cells with some subtypes being restricted to a single brain region while others were found globally across the brain. Global stem cell states are linked to neurogenic differentiation with different states being involved in proliferative and non proliferative differentiation. Our work reveals principles of adult stem cell organization and establishes a resource for functional manipulation of neural stem cell subtypes. Overall design: Single cell RNA seq of adult zebrafish brain performed using 10x Genomics Gene Expression protocols to explore transcriptomic diversity of adult cell types. The samples were prepared either from whole brain FACS sorted cells GFP positive cells from gfap:GFP line or a dissected single brain region as indicated in the sample title. CRISPR based lineage tracing was performed for a subset of samples to dissect developmental lineage relationships between cells. ScSLAM seq was performed in combination with Notch pathway inhibition to assess response of stem cells to perturbation.   pubmed:38365956   Brain 22 telencephalon control scSLAMseq GSM7875185   source name:adult brain|tissue:adult brain|tissue region:telencephalon|cell type:mixed tissue dissociation|genotype:wildtype|treatment:Control DMSO|geo loc name:missing|collection date:missing Brain 22 telencephalon control scSLAMseq The transcriptome libraries were demultiplexed and mapped with CellRanger 6.1.1. In order to reduce batch effect the gene expression matrices were processed with the SoupX tool Young & Behjati 2020 for removing ambient RNA contamination. A subset of libraries was also mapped with Velocyto v0.17.17 La Manno et al 2018 to create count matrices for unspliced and spliced molecules. The targeted lineage tracing libraries were aligned using bwa mem3 v.0.7.12 to individual references of endogenous genes used in the lineage tracing experiment actb1 actb2 cfl1 cirbpb rpl39 and ube2e1. The scars were filtered with a custom pipeline to eliminate sequences that originate from PCR or sequencing errors. For custom code and further downstream analysis see: https://github.com/nimitic/radial glia. Assembly: GRCz11 Supplementary files format and content: Tab separated barcode files and matrix files along with the tab separated gene file supplementary file are the output of CellRanger mapping further processed by the SoupX package for ambient RNA removal. Supplementary files format and content: Loom files are the output of velocyto mapping distinguishing spliced and unspliced mRNAs. Supplementary files format and content: filtered scars.csv files contain the output of a custom scar filtering pipeline. Library strategy: scSLAM seq adult brain Control for Notch inhibition: zebrafish were incubated in a water bath containing system water with 1:200 diluted DMSO for 48 hours. The samples were prepared according to a scSLAM seq protocol adapted from Neuschulz et al 2023. Briefly: in order to label nascent transcripts 200 mM 4sU was delivered to fish brains by intraventricular injection 6 hours prior to sample collection. The brains were then collected and a single cell suspension was prepared by papain dissociation. The resulting cell suspension was fixed in 80% methanol and a conversion of 4sU using iodoactamide adding 111 µl 100 mM IAA to 800 µl fixed sample was done overnight. The following day the reaction was quanched with a quenching buffer containing 100 mM DTT post which the sample was washed with a wash buffer and filtered though a 35 µm filter. The sample was then loaded on a 10X Chromium Controller and processed according to the standard scRNA seq protocol. Adult zebrafish brain tissue was extracted optionally dissected to select a specific region of interest telencephalon diencephalon mesencephalon or rhombencephalon and then dissociated with a trypsin protocol. Briefly the samples were incubated in 750 µl of HBSS glucose solution and dissocated with 15 µl of 0.05% trypsin EDTA Gibco for 30 minutes at 37°C with intermittent mixing. The disociation was stopped by addition of 750 µl of a BSA EBSS HEPES solution. Further sample handling steps were carried out on ice and for centrifugation at 4°C. The sample was filtered through a 100 µm filter washed with cold HBSS and resuspended in 100 200 µl HBSS with 0.05% BSA then filtered through a 35 µm filter and loaded on the 10x Chromium Controller. For the scSLAM seq samples a papain dissociation protocol Worthington kit according to manufecturer's instructions was used followed by an adapted scSLAM seq protocol for labeling nascent transcripts see sample specific entry. The standard transcriptome libraries were constructed according to the manufacturer's protocol 10X Genomics. For targeted libraries of scars used for lineage tracing the cDNA obtained during the 10x protocol was used to set up individual PCR reactions …   tissue:adult brain|tissue region:telencephalon|cell type:mixed tissue dissociation|genotype:wildtype|treatment:Control DMSO GSM7875185 GSM7875185: Brain 22 telencephalon control scSLAMseq; Danio rerio; OTHER GSM7875185 r1 GSM7875185 1 The samples were prepared according to a scSLAM seq protocol adapted from Neuschulz et al 2023. Briefly: in order to label nascent transcripts 200 mM 4sU was delivered to fish brains by intraventricular injection 6 hours prior to sample collection. The brains were then collected and a single cell suspension was prepared by papain dissociation. The resulting cell suspension was fixed in 80% methanol and a conversion of 4sU using iodoactamide adding 111 µl 100 mM IAA to 800 µl fixed sample was done overnight. The following day the reaction was quanched with a quenching buffer containing 100 mM DTT post which the sample was washed with a wash buffer and filtered though a 35 µm filter. The sample was then loaded on a 10X Chromium Controller and processed according to the standard scRNA seq protocol. Adult zebrafish brain tissue was extracted optionally dissected to select a specific region of interest telencephalon diencephalon mesencephalon or rhombencephalon and then dissociated with a trypsin protocol. Briefly the samples were incubated in 750 µl of HBSS glucose solution and dissocated with 15 µl of 0.05% trypsin EDTA Gibco for 30 minutes at 37°C with intermittent mixing. The disociation was stopped by addition of 750 µl of a BSA EBSS HEPES solution. Further sample handling steps were carried out on ice and for centrifugation at 4°C. The sample was filtered through a 100 µm filter washed with cold HBSS and resuspended in 100 200 µl HBSS with 0.05% BSA then filtered through a 35 µm filter and loaded on the 10x Chromium Controller. For the scSLAM seq samples a papain dissociation protocol Worthington kit according to manufecturer's instructions was used followed by an adapted scSLAM seq protocol for labeling nascent transcripts see sample specific entry. The standard transcriptome libraries were constructed according to the manufacturer's protocol 10X Genomics. For targeted libraries of scars used for lineage tracing the cDNA obtained during the 10x protocol was used to set up individual PCR reactions …   OTHER TRANSCRIPTOMIC other PAIRED ILLUMINA Illumina NovaSeq 6000   SRP469552   loader:fastq load.py b22_ni_con_R1.fastq.gz b22_ni_con_R2.fastq.gz fastq fastq 59793114490.0 259970063.0 GSM7875185 r1 0:28 1:202 A:18600519530;C:12714987967;G:14147370465;T:14316863915;N:13372613 28 202     18600519530 12714987967 14147370465 14316863915 13372613 SRX22323916 SRS19374445 SRA1743007 Junker Lab, Berlin Institute for Medical Systems Biology, Max-Delbrück-Center for Molecular Medicine Junker Lab, Berlin Institute for Medical Systems Biology, Max-Delbrück-Center for Molecular Medicine 2 0.01402 0.79448 0.00498 0.11405 0.99074 0.8518 0.39874 0.67082 28 202 T B sc-like readlen illumina novaseq_era unknown other unknown sc single_cell_droplet 10x   Germany 2023-10-31 Adult Adult Brain Nervous System
28754 28754 SRR26623291 SRX22323897 SRS19374426 SRP469552 PRJNA1034159 Dissecting the spatiotemporal diversity of adult neural stem cells GSE246714 Other Adult stem cells are important for tissue turnover and regeneration. However in most adult systems it remains elusive how stem cells assume different functional states and support spatially patterned tissue architecture. Here we dissected the diversity of neural stem cells in the adult zebrafish brain an organ that is characterized by pronounced zonation and high regenerative capacity. We combined single cell transcriptomics of dissected brain regions with massively parallel lineage tracing and in vivo RNA metabolic labeling to analyze regulation of neural stem cells in space and time. We detected a large diversity of neural stem cells with some subtypes being restricted to a single brain region while others were found globally across the brain. Global stem cell states are linked to neurogenic differentiation with different states being involved in proliferative and non proliferative differentiation. Our work reveals principles of adult stem cell organization and establishes a resource for functional manipulation of neural stem cell subtypes. Overall design: Single cell RNA seq of adult zebrafish brain performed using 10x Genomics Gene Expression protocols to explore transcriptomic diversity of adult cell types. The samples were prepared either from whole brain FACS sorted cells GFP positive cells from gfap:GFP line or a dissected single brain region as indicated in the sample title. CRISPR based lineage tracing was performed for a subset of samples to dissect developmental lineage relationships between cells. ScSLAM seq was performed in combination with Notch pathway inhibition to assess response of stem cells to perturbation.   pubmed:38365956   Lineage tracing rep2 ube2e1 scars GSM7875196   source name:adult brain|tissue:adult brain|cell type:mixed tissue dissociation|genotype:Tg[ubi:zebrabow M]|geo loc name:missing|collection date:missing Lineage tracing rep2 ube2e1 scars The transcriptome libraries were demultiplexed and mapped with CellRanger 6.1.1. In order to reduce batch effect the gene expression matrices were processed with the SoupX tool Young & Behjati 2020 for removing ambient RNA contamination. A subset of libraries was also mapped with Velocyto v0.17.17 La Manno et al 2018 to create count matrices for unspliced and spliced molecules. The targeted lineage tracing libraries were aligned using bwa mem3 v.0.7.12 to individual references of endogenous genes used in the lineage tracing experiment actb1 actb2 cfl1 cirbpb rpl39 and ube2e1. The scars were filtered with a custom pipeline to eliminate sequences that originate from PCR or sequencing errors. For custom code and further downstream analysis see: https://github.com/nimitic/radial glia. Assembly: GRCz11 Supplementary files format and content: Tab separated barcode files and matrix files along with the tab separated gene file supplementary file are the output of CellRanger mapping further processed by the SoupX package for ambient RNA removal. Supplementary files format and content: Loom files are the output of velocyto mapping distinguishing spliced and unspliced mRNAs. Supplementary files format and content: filtered scars.csv files contain the output of a custom scar filtering pipeline. adult brain   Adult zebrafish brain tissue was extracted optionally dissected to select a specific region of interest telencephalon diencephalon mesencephalon or rhombencephalon and then dissociated with a trypsin protocol. Briefly the samples were incubated in 750 µl of HBSS glucose solution and dissocated with 15 µl of 0.05% trypsin EDTA Gibco for 30 minutes at 37°C with intermittent mixing. The disociation was stopped by addition of 750 µl of a BSA EBSS HEPES solution. Further sample handling steps were carried out on ice and for centrifugation at 4°C. The sample was filtered through a 100 µm filter washed with cold HBSS and resuspended in 100 200 µl HBSS with 0.05% BSA then filtered through a 35 µm filter and loaded on the 10x Chromium Controller. For the scSLAM seq samples a papain dissociation protocol Worthington kit according to manufecturer's instructions was used followed by an adapted scSLAM seq protocol for labeling nascent transcripts see sample specific entry. The standard transcriptome libraries were constructed according to the manufacturer's protocol 10X Genomics. For targeted libraries of scars used for lineage tracing the cDNA obtained during the 10x protocol was used to set up individual PCR reactions for each target specifically amplifying the sgRNA target site. The libraries for targeted PCR were constructed with primers compatible with the 10X Genomics kit so they could be sequenced on the same platform.   tissue:adult brain|cell type:mixed tissue dissociation|genotype:Tg[ubi:zebrabow M] GSM7875196 GSM7875196: Lineage tracing rep2 ube2e1 scars; Danio rerio; OTHER GSM7875196 r1 GSM7875196 1 Adult zebrafish brain tissue was extracted optionally dissected to select a specific region of interest telencephalon diencephalon mesencephalon or rhombencephalon and then dissociated with a trypsin protocol. Briefly the samples were incubated in 750 µl of HBSS glucose solution and dissocated with 15 µl of 0.05% trypsin EDTA Gibco for 30 minutes at 37°C with intermittent mixing. The disociation was stopped by addition of 750 µl of a BSA EBSS HEPES solution. Further sample handling steps were carried out on ice and for centrifugation at 4°C. The sample was filtered through a 100 µm filter washed with cold HBSS and resuspended in 100 200 µl HBSS with 0.05% BSA then filtered through a 35 µm filter and loaded on the 10x Chromium Controller. For the scSLAM seq samples a papain dissociation protocol Worthington kit according to manufecturer's instructions was used followed by an adapted scSLAM seq protocol for labeling nascent transcripts see sample specific entry. The standard transcriptome libraries were constructed according to the manufacturer's protocol 10X Genomics. For targeted libraries of scars used for lineage tracing the cDNA obtained during the 10x protocol was used to set up individual PCR reactions for each target specifically amplifying the sgRNA target site. The libraries for targeted PCR were constructed with primers compatible with the 10X Genomics kit so they could be sequenced on the same platform.   OTHER TRANSCRIPTOMIC other PAIRED ILLUMINA Illumina NovaSeq 6000   SRP469552   loader:fastq load.py lin2_ube2e1_scar_R1.fastq.gz lin2_ube2e1_scar_R2.fastq.gz fastq fastq 213092161.0 1190459.0 GSM7875196 r1                       SRX22323897 SRS19374426 SRA1743007 Junker Lab, Berlin Institute for Medical Systems Biology, Max-Delbrück-Center for Molecular Medicine Junker Lab, Berlin Institute for Medical Systems Biology, Max-Delbrück-Center for Molecular Medicine 2 0.00752 0.90776 0.00286 0.01739 0.99346 0.95444 0.46002 0.96817 28 151 T B sc-like readlen illumina novaseq_era unknown other unknown sc single_cell_droplet 10x   Germany 2023-10-31 Adult Adult Brain Nervous System
28755 28755 SRR26623292 SRX22323896 SRS19374425 SRP469552 PRJNA1034159 Dissecting the spatiotemporal diversity of adult neural stem cells GSE246714 Other Adult stem cells are important for tissue turnover and regeneration. However in most adult systems it remains elusive how stem cells assume different functional states and support spatially patterned tissue architecture. Here we dissected the diversity of neural stem cells in the adult zebrafish brain an organ that is characterized by pronounced zonation and high regenerative capacity. We combined single cell transcriptomics of dissected brain regions with massively parallel lineage tracing and in vivo RNA metabolic labeling to analyze regulation of neural stem cells in space and time. We detected a large diversity of neural stem cells with some subtypes being restricted to a single brain region while others were found globally across the brain. Global stem cell states are linked to neurogenic differentiation with different states being involved in proliferative and non proliferative differentiation. Our work reveals principles of adult stem cell organization and establishes a resource for functional manipulation of neural stem cell subtypes. Overall design: Single cell RNA seq of adult zebrafish brain performed using 10x Genomics Gene Expression protocols to explore transcriptomic diversity of adult cell types. The samples were prepared either from whole brain FACS sorted cells GFP positive cells from gfap:GFP line or a dissected single brain region as indicated in the sample title. CRISPR based lineage tracing was performed for a subset of samples to dissect developmental lineage relationships between cells. ScSLAM seq was performed in combination with Notch pathway inhibition to assess response of stem cells to perturbation.   pubmed:38365956   Lineage tracing rep2 rpl39 scars GSM7875195   source name:adult brain|tissue:adult brain|cell type:mixed tissue dissociation|genotype:Tg[ubi:zebrabow M]|geo loc name:missing|collection date:missing Lineage tracing rep2 rpl39 scars The transcriptome libraries were demultiplexed and mapped with CellRanger 6.1.1. In order to reduce batch effect the gene expression matrices were processed with the SoupX tool Young & Behjati 2020 for removing ambient RNA contamination. A subset of libraries was also mapped with Velocyto v0.17.17 La Manno et al 2018 to create count matrices for unspliced and spliced molecules. The targeted lineage tracing libraries were aligned using bwa mem3 v.0.7.12 to individual references of endogenous genes used in the lineage tracing experiment actb1 actb2 cfl1 cirbpb rpl39 and ube2e1. The scars were filtered with a custom pipeline to eliminate sequences that originate from PCR or sequencing errors. For custom code and further downstream analysis see: https://github.com/nimitic/radial glia. Assembly: GRCz11 Supplementary files format and content: Tab separated barcode files and matrix files along with the tab separated gene file supplementary file are the output of CellRanger mapping further processed by the SoupX package for ambient RNA removal. Supplementary files format and content: Loom files are the output of velocyto mapping distinguishing spliced and unspliced mRNAs. Supplementary files format and content: filtered scars.csv files contain the output of a custom scar filtering pipeline. adult brain   Adult zebrafish brain tissue was extracted optionally dissected to select a specific region of interest telencephalon diencephalon mesencephalon or rhombencephalon and then dissociated with a trypsin protocol. Briefly the samples were incubated in 750 µl of HBSS glucose solution and dissocated with 15 µl of 0.05% trypsin EDTA Gibco for 30 minutes at 37°C with intermittent mixing. The disociation was stopped by addition of 750 µl of a BSA EBSS HEPES solution. Further sample handling steps were carried out on ice and for centrifugation at 4°C. The sample was filtered through a 100 µm filter washed with cold HBSS and resuspended in 100 200 µl HBSS with 0.05% BSA then filtered through a 35 µm filter and loaded on the 10x Chromium Controller. For the scSLAM seq samples a papain dissociation protocol Worthington kit according to manufecturer's instructions was used followed by an adapted scSLAM seq protocol for labeling nascent transcripts see sample specific entry. The standard transcriptome libraries were constructed according to the manufacturer's protocol 10X Genomics. For targeted libraries of scars used for lineage tracing the cDNA obtained during the 10x protocol was used to set up individual PCR reactions for each target specifically amplifying the sgRNA target site. The libraries for targeted PCR were constructed with primers compatible with the 10X Genomics kit so they could be sequenced on the same platform.   tissue:adult brain|cell type:mixed tissue dissociation|genotype:Tg[ubi:zebrabow M] GSM7875195 GSM7875195: Lineage tracing rep2 rpl39 scars; Danio rerio; OTHER GSM7875195 r1 GSM7875195 1 Adult zebrafish brain tissue was extracted optionally dissected to select a specific region of interest telencephalon diencephalon mesencephalon or rhombencephalon and then dissociated with a trypsin protocol. Briefly the samples were incubated in 750 µl of HBSS glucose solution and dissocated with 15 µl of 0.05% trypsin EDTA Gibco for 30 minutes at 37°C with intermittent mixing. The disociation was stopped by addition of 750 µl of a BSA EBSS HEPES solution. Further sample handling steps were carried out on ice and for centrifugation at 4°C. The sample was filtered through a 100 µm filter washed with cold HBSS and resuspended in 100 200 µl HBSS with 0.05% BSA then filtered through a 35 µm filter and loaded on the 10x Chromium Controller. For the scSLAM seq samples a papain dissociation protocol Worthington kit according to manufecturer's instructions was used followed by an adapted scSLAM seq protocol for labeling nascent transcripts see sample specific entry. The standard transcriptome libraries were constructed according to the manufacturer's protocol 10X Genomics. For targeted libraries of scars used for lineage tracing the cDNA obtained during the 10x protocol was used to set up individual PCR reactions for each target specifically amplifying the sgRNA target site. The libraries for targeted PCR were constructed with primers compatible with the 10X Genomics kit so they could be sequenced on the same platform.   OTHER TRANSCRIPTOMIC other PAIRED ILLUMINA Illumina NovaSeq 6000   SRP469552   loader:fastq load.py lin2_rpl39_scar_R2.fastq.gz lin2_rpl39_scar_R1.fastq.gz fastq fastq 1023403502.0 5717338.0 GSM7875195 r1                       SRX22323896 SRS19374425 SRA1743007 Junker Lab, Berlin Institute for Medical Systems Biology, Max-Delbrück-Center for Molecular Medicine Junker Lab, Berlin Institute for Medical Systems Biology, Max-Delbrück-Center for Molecular Medicine 2 0.00086 0.83744 0.0003 0.00337 0.99845 0.97057 0.26605 0.40241 28 151 T B sc-like readlen illumina novaseq_era unknown other unknown sc single_cell_droplet 10x   Germany 2023-10-31 Adult Adult Brain Nervous System
28756 28756 SRR26623293 SRX22323895 SRS19374424 SRP469552 PRJNA1034159 Dissecting the spatiotemporal diversity of adult neural stem cells GSE246714 Other Adult stem cells are important for tissue turnover and regeneration. However in most adult systems it remains elusive how stem cells assume different functional states and support spatially patterned tissue architecture. Here we dissected the diversity of neural stem cells in the adult zebrafish brain an organ that is characterized by pronounced zonation and high regenerative capacity. We combined single cell transcriptomics of dissected brain regions with massively parallel lineage tracing and in vivo RNA metabolic labeling to analyze regulation of neural stem cells in space and time. We detected a large diversity of neural stem cells with some subtypes being restricted to a single brain region while others were found globally across the brain. Global stem cell states are linked to neurogenic differentiation with different states being involved in proliferative and non proliferative differentiation. Our work reveals principles of adult stem cell organization and establishes a resource for functional manipulation of neural stem cell subtypes. Overall design: Single cell RNA seq of adult zebrafish brain performed using 10x Genomics Gene Expression protocols to explore transcriptomic diversity of adult cell types. The samples were prepared either from whole brain FACS sorted cells GFP positive cells from gfap:GFP line or a dissected single brain region as indicated in the sample title. CRISPR based lineage tracing was performed for a subset of samples to dissect developmental lineage relationships between cells. ScSLAM seq was performed in combination with Notch pathway inhibition to assess response of stem cells to perturbation.   pubmed:38365956   Lineage tracing rep2 cirbpb scars GSM7875194   source name:adult brain|tissue:adult brain|cell type:mixed tissue dissociation|genotype:Tg[ubi:zebrabow M]|geo loc name:missing|collection date:missing Lineage tracing rep2 cirbpb scars The transcriptome libraries were demultiplexed and mapped with CellRanger 6.1.1. In order to reduce batch effect the gene expression matrices were processed with the SoupX tool Young & Behjati 2020 for removing ambient RNA contamination. A subset of libraries was also mapped with Velocyto v0.17.17 La Manno et al 2018 to create count matrices for unspliced and spliced molecules. The targeted lineage tracing libraries were aligned using bwa mem3 v.0.7.12 to individual references of endogenous genes used in the lineage tracing experiment actb1 actb2 cfl1 cirbpb rpl39 and ube2e1. The scars were filtered with a custom pipeline to eliminate sequences that originate from PCR or sequencing errors. For custom code and further downstream analysis see: https://github.com/nimitic/radial glia. Assembly: GRCz11 Supplementary files format and content: Tab separated barcode files and matrix files along with the tab separated gene file supplementary file are the output of CellRanger mapping further processed by the SoupX package for ambient RNA removal. Supplementary files format and content: Loom files are the output of velocyto mapping distinguishing spliced and unspliced mRNAs. Supplementary files format and content: filtered scars.csv files contain the output of a custom scar filtering pipeline. adult brain   Adult zebrafish brain tissue was extracted optionally dissected to select a specific region of interest telencephalon diencephalon mesencephalon or rhombencephalon and then dissociated with a trypsin protocol. Briefly the samples were incubated in 750 µl of HBSS glucose solution and dissocated with 15 µl of 0.05% trypsin EDTA Gibco for 30 minutes at 37°C with intermittent mixing. The disociation was stopped by addition of 750 µl of a BSA EBSS HEPES solution. Further sample handling steps were carried out on ice and for centrifugation at 4°C. The sample was filtered through a 100 µm filter washed with cold HBSS and resuspended in 100 200 µl HBSS with 0.05% BSA then filtered through a 35 µm filter and loaded on the 10x Chromium Controller. For the scSLAM seq samples a papain dissociation protocol Worthington kit according to manufecturer's instructions was used followed by an adapted scSLAM seq protocol for labeling nascent transcripts see sample specific entry. The standard transcriptome libraries were constructed according to the manufacturer's protocol 10X Genomics. For targeted libraries of scars used for lineage tracing the cDNA obtained during the 10x protocol was used to set up individual PCR reactions for each target specifically amplifying the sgRNA target site. The libraries for targeted PCR were constructed with primers compatible with the 10X Genomics kit so they could be sequenced on the same platform.   tissue:adult brain|cell type:mixed tissue dissociation|genotype:Tg[ubi:zebrabow M] GSM7875194 GSM7875194: Lineage tracing rep2 cirbpb scars; Danio rerio; OTHER GSM7875194 r1 GSM7875194 1 Adult zebrafish brain tissue was extracted optionally dissected to select a specific region of interest telencephalon diencephalon mesencephalon or rhombencephalon and then dissociated with a trypsin protocol. Briefly the samples were incubated in 750 µl of HBSS glucose solution and dissocated with 15 µl of 0.05% trypsin EDTA Gibco for 30 minutes at 37°C with intermittent mixing. The disociation was stopped by addition of 750 µl of a BSA EBSS HEPES solution. Further sample handling steps were carried out on ice and for centrifugation at 4°C. The sample was filtered through a 100 µm filter washed with cold HBSS and resuspended in 100 200 µl HBSS with 0.05% BSA then filtered through a 35 µm filter and loaded on the 10x Chromium Controller. For the scSLAM seq samples a papain dissociation protocol Worthington kit according to manufecturer's instructions was used followed by an adapted scSLAM seq protocol for labeling nascent transcripts see sample specific entry. The standard transcriptome libraries were constructed according to the manufacturer's protocol 10X Genomics. For targeted libraries of scars used for lineage tracing the cDNA obtained during the 10x protocol was used to set up individual PCR reactions for each target specifically amplifying the sgRNA target site. The libraries for targeted PCR were constructed with primers compatible with the 10X Genomics kit so they could be sequenced on the same platform.   OTHER TRANSCRIPTOMIC other PAIRED ILLUMINA Illumina NovaSeq 6000   SRP469552   loader:fastq load.py lin2_cirbpb_scar_R1.fastq.gz lin2_cirbpb_scar_R2.fastq.gz fastq fastq 276120388.0 1542572.0 GSM7875194 r1                       SRX22323895 SRS19374424 SRA1743007 Junker Lab, Berlin Institute for Medical Systems Biology, Max-Delbrück-Center for Molecular Medicine Junker Lab, Berlin Institute for Medical Systems Biology, Max-Delbrück-Center for Molecular Medicine 2 0.00206 0.80328 0.00078 0.00662 0.9975 0.98039 0.55421 0.0258 28 151 T B sc-like readlen illumina novaseq_era unknown other unknown sc single_cell_droplet 10x   Germany 2023-10-31 Adult Adult Brain Nervous System
28757 28757 SRR26623294 SRX22323894 SRS19374423 SRP469552 PRJNA1034159 Dissecting the spatiotemporal diversity of adult neural stem cells GSE246714 Other Adult stem cells are important for tissue turnover and regeneration. However in most adult systems it remains elusive how stem cells assume different functional states and support spatially patterned tissue architecture. Here we dissected the diversity of neural stem cells in the adult zebrafish brain an organ that is characterized by pronounced zonation and high regenerative capacity. We combined single cell transcriptomics of dissected brain regions with massively parallel lineage tracing and in vivo RNA metabolic labeling to analyze regulation of neural stem cells in space and time. We detected a large diversity of neural stem cells with some subtypes being restricted to a single brain region while others were found globally across the brain. Global stem cell states are linked to neurogenic differentiation with different states being involved in proliferative and non proliferative differentiation. Our work reveals principles of adult stem cell organization and establishes a resource for functional manipulation of neural stem cell subtypes. Overall design: Single cell RNA seq of adult zebrafish brain performed using 10x Genomics Gene Expression protocols to explore transcriptomic diversity of adult cell types. The samples were prepared either from whole brain FACS sorted cells GFP positive cells from gfap:GFP line or a dissected single brain region as indicated in the sample title. CRISPR based lineage tracing was performed for a subset of samples to dissect developmental lineage relationships between cells. ScSLAM seq was performed in combination with Notch pathway inhibition to assess response of stem cells to perturbation.   pubmed:38365956   Lineage tracing rep2 cfl1 scars GSM7875193   source name:adult brain|tissue:adult brain|cell type:mixed tissue dissociation|genotype:Tg[ubi:zebrabow M]|geo loc name:missing|collection date:missing Lineage tracing rep2 cfl1 scars The transcriptome libraries were demultiplexed and mapped with CellRanger 6.1.1. In order to reduce batch effect the gene expression matrices were processed with the SoupX tool Young & Behjati 2020 for removing ambient RNA contamination. A subset of libraries was also mapped with Velocyto v0.17.17 La Manno et al 2018 to create count matrices for unspliced and spliced molecules. The targeted lineage tracing libraries were aligned using bwa mem3 v.0.7.12 to individual references of endogenous genes used in the lineage tracing experiment actb1 actb2 cfl1 cirbpb rpl39 and ube2e1. The scars were filtered with a custom pipeline to eliminate sequences that originate from PCR or sequencing errors. For custom code and further downstream analysis see: https://github.com/nimitic/radial glia. Assembly: GRCz11 Supplementary files format and content: Tab separated barcode files and matrix files along with the tab separated gene file supplementary file are the output of CellRanger mapping further processed by the SoupX package for ambient RNA removal. Supplementary files format and content: Loom files are the output of velocyto mapping distinguishing spliced and unspliced mRNAs. Supplementary files format and content: filtered scars.csv files contain the output of a custom scar filtering pipeline. adult brain   Adult zebrafish brain tissue was extracted optionally dissected to select a specific region of interest telencephalon diencephalon mesencephalon or rhombencephalon and then dissociated with a trypsin protocol. Briefly the samples were incubated in 750 µl of HBSS glucose solution and dissocated with 15 µl of 0.05% trypsin EDTA Gibco for 30 minutes at 37°C with intermittent mixing. The disociation was stopped by addition of 750 µl of a BSA EBSS HEPES solution. Further sample handling steps were carried out on ice and for centrifugation at 4°C. The sample was filtered through a 100 µm filter washed with cold HBSS and resuspended in 100 200 µl HBSS with 0.05% BSA then filtered through a 35 µm filter and loaded on the 10x Chromium Controller. For the scSLAM seq samples a papain dissociation protocol Worthington kit according to manufecturer's instructions was used followed by an adapted scSLAM seq protocol for labeling nascent transcripts see sample specific entry. The standard transcriptome libraries were constructed according to the manufacturer's protocol 10X Genomics. For targeted libraries of scars used for lineage tracing the cDNA obtained during the 10x protocol was used to set up individual PCR reactions for each target specifically amplifying the sgRNA target site. The libraries for targeted PCR were constructed with primers compatible with the 10X Genomics kit so they could be sequenced on the same platform.   tissue:adult brain|cell type:mixed tissue dissociation|genotype:Tg[ubi:zebrabow M] GSM7875193 GSM7875193: Lineage tracing rep2 cfl1 scars; Danio rerio; OTHER GSM7875193 r1 GSM7875193 1 Adult zebrafish brain tissue was extracted optionally dissected to select a specific region of interest telencephalon diencephalon mesencephalon or rhombencephalon and then dissociated with a trypsin protocol. Briefly the samples were incubated in 750 µl of HBSS glucose solution and dissocated with 15 µl of 0.05% trypsin EDTA Gibco for 30 minutes at 37°C with intermittent mixing. The disociation was stopped by addition of 750 µl of a BSA EBSS HEPES solution. Further sample handling steps were carried out on ice and for centrifugation at 4°C. The sample was filtered through a 100 µm filter washed with cold HBSS and resuspended in 100 200 µl HBSS with 0.05% BSA then filtered through a 35 µm filter and loaded on the 10x Chromium Controller. For the scSLAM seq samples a papain dissociation protocol Worthington kit according to manufecturer's instructions was used followed by an adapted scSLAM seq protocol for labeling nascent transcripts see sample specific entry. The standard transcriptome libraries were constructed according to the manufacturer's protocol 10X Genomics. For targeted libraries of scars used for lineage tracing the cDNA obtained during the 10x protocol was used to set up individual PCR reactions for each target specifically amplifying the sgRNA target site. The libraries for targeted PCR were constructed with primers compatible with the 10X Genomics kit so they could be sequenced on the same platform.   OTHER TRANSCRIPTOMIC other PAIRED ILLUMINA Illumina NovaSeq 6000   SRP469552   loader:fastq load.py lin2_cfl1_scar_R1.fastq.gz lin2_cfl1_scar_R2.fastq.gz fastq fastq 791318009.0 4420771.0 GSM7875193 r1                       SRX22323894 SRS19374423 SRA1743007 Junker Lab, Berlin Institute for Medical Systems Biology, Max-Delbrück-Center for Molecular Medicine Junker Lab, Berlin Institute for Medical Systems Biology, Max-Delbrück-Center for Molecular Medicine 2 0.00067 0.46079 0.00055 0.16353 0.99965 0.99332 0.52631 0.44939 28 151 T B sc-like readlen illumina novaseq_era unknown other unknown sc single_cell_droplet 10x   Germany 2023-10-31 Adult Adult Brain Nervous System
28758 28758 SRR26623295 SRX22323893 SRS19374421 SRP469552 PRJNA1034159 Dissecting the spatiotemporal diversity of adult neural stem cells GSE246714 Other Adult stem cells are important for tissue turnover and regeneration. However in most adult systems it remains elusive how stem cells assume different functional states and support spatially patterned tissue architecture. Here we dissected the diversity of neural stem cells in the adult zebrafish brain an organ that is characterized by pronounced zonation and high regenerative capacity. We combined single cell transcriptomics of dissected brain regions with massively parallel lineage tracing and in vivo RNA metabolic labeling to analyze regulation of neural stem cells in space and time. We detected a large diversity of neural stem cells with some subtypes being restricted to a single brain region while others were found globally across the brain. Global stem cell states are linked to neurogenic differentiation with different states being involved in proliferative and non proliferative differentiation. Our work reveals principles of adult stem cell organization and establishes a resource for functional manipulation of neural stem cell subtypes. Overall design: Single cell RNA seq of adult zebrafish brain performed using 10x Genomics Gene Expression protocols to explore transcriptomic diversity of adult cell types. The samples were prepared either from whole brain FACS sorted cells GFP positive cells from gfap:GFP line or a dissected single brain region as indicated in the sample title. CRISPR based lineage tracing was performed for a subset of samples to dissect developmental lineage relationships between cells. ScSLAM seq was performed in combination with Notch pathway inhibition to assess response of stem cells to perturbation.   pubmed:38365956   Lineage tracing rep1 ube2e1 scars GSM7875192   source name:adult brain|tissue:adult brain|cell type:mixed tissue dissociation|genotype:Tg[ubi:zebrabow M]|geo loc name:missing|collection date:missing Lineage tracing rep1 ube2e1 scars The transcriptome libraries were demultiplexed and mapped with CellRanger 6.1.1. In order to reduce batch effect the gene expression matrices were processed with the SoupX tool Young & Behjati 2020 for removing ambient RNA contamination. A subset of libraries was also mapped with Velocyto v0.17.17 La Manno et al 2018 to create count matrices for unspliced and spliced molecules. The targeted lineage tracing libraries were aligned using bwa mem3 v.0.7.12 to individual references of endogenous genes used in the lineage tracing experiment actb1 actb2 cfl1 cirbpb rpl39 and ube2e1. The scars were filtered with a custom pipeline to eliminate sequences that originate from PCR or sequencing errors. For custom code and further downstream analysis see: https://github.com/nimitic/radial glia. Assembly: GRCz11 Supplementary files format and content: Tab separated barcode files and matrix files along with the tab separated gene file supplementary file are the output of CellRanger mapping further processed by the SoupX package for ambient RNA removal. Supplementary files format and content: Loom files are the output of velocyto mapping distinguishing spliced and unspliced mRNAs. Supplementary files format and content: filtered scars.csv files contain the output of a custom scar filtering pipeline. adult brain   Adult zebrafish brain tissue was extracted optionally dissected to select a specific region of interest telencephalon diencephalon mesencephalon or rhombencephalon and then dissociated with a trypsin protocol. Briefly the samples were incubated in 750 µl of HBSS glucose solution and dissocated with 15 µl of 0.05% trypsin EDTA Gibco for 30 minutes at 37°C with intermittent mixing. The disociation was stopped by addition of 750 µl of a BSA EBSS HEPES solution. Further sample handling steps were carried out on ice and for centrifugation at 4°C. The sample was filtered through a 100 µm filter washed with cold HBSS and resuspended in 100 200 µl HBSS with 0.05% BSA then filtered through a 35 µm filter and loaded on the 10x Chromium Controller. For the scSLAM seq samples a papain dissociation protocol Worthington kit according to manufecturer's instructions was used followed by an adapted scSLAM seq protocol for labeling nascent transcripts see sample specific entry. The standard transcriptome libraries were constructed according to the manufacturer's protocol 10X Genomics. For targeted libraries of scars used for lineage tracing the cDNA obtained during the 10x protocol was used to set up individual PCR reactions for each target specifically amplifying the sgRNA target site. The libraries for targeted PCR were constructed with primers compatible with the 10X Genomics kit so they could be sequenced on the same platform.   tissue:adult brain|cell type:mixed tissue dissociation|genotype:Tg[ubi:zebrabow M] GSM7875192 GSM7875192: Lineage tracing rep1 ube2e1 scars; Danio rerio; OTHER GSM7875192 r1 GSM7875192 1 Adult zebrafish brain tissue was extracted optionally dissected to select a specific region of interest telencephalon diencephalon mesencephalon or rhombencephalon and then dissociated with a trypsin protocol. Briefly the samples were incubated in 750 µl of HBSS glucose solution and dissocated with 15 µl of 0.05% trypsin EDTA Gibco for 30 minutes at 37°C with intermittent mixing. The disociation was stopped by addition of 750 µl of a BSA EBSS HEPES solution. Further sample handling steps were carried out on ice and for centrifugation at 4°C. The sample was filtered through a 100 µm filter washed with cold HBSS and resuspended in 100 200 µl HBSS with 0.05% BSA then filtered through a 35 µm filter and loaded on the 10x Chromium Controller. For the scSLAM seq samples a papain dissociation protocol Worthington kit according to manufecturer's instructions was used followed by an adapted scSLAM seq protocol for labeling nascent transcripts see sample specific entry. The standard transcriptome libraries were constructed according to the manufacturer's protocol 10X Genomics. For targeted libraries of scars used for lineage tracing the cDNA obtained during the 10x protocol was used to set up individual PCR reactions for each target specifically amplifying the sgRNA target site. The libraries for targeted PCR were constructed with primers compatible with the 10X Genomics kit so they could be sequenced on the same platform.   OTHER TRANSCRIPTOMIC other PAIRED ILLUMINA NextSeq 500   SRP469552   loader:fastq load.py lin1_ube2e1_scar_R1.fastq.gz lin1_ube2e1_scar_R2.fastq.gz fastq fastq 99407618.0 601781.0 GSM7875192 r1                       SRX22323893 SRS19374421 SRA1743007 Junker Lab, Berlin Institute for Medical Systems Biology, Max-Delbrück-Center for Molecular Medicine Junker Lab, Berlin Institute for Medical Systems Biology, Max-Delbrück-Center for Molecular Medicine 2 0.00657 0.84532 0.00205 0.03049 0.99537 0.95286 0.43306 0.19203 28 150 T B sc-like readlen illumina nextseq unknown other unknown sc single_cell_droplet 10x   Germany 2023-10-31 Adult Adult Brain Nervous System
28759 28759 SRR26623296 SRX22323892 SRS19374422 SRP469552 PRJNA1034159 Dissecting the spatiotemporal diversity of adult neural stem cells GSE246714 Other Adult stem cells are important for tissue turnover and regeneration. However in most adult systems it remains elusive how stem cells assume different functional states and support spatially patterned tissue architecture. Here we dissected the diversity of neural stem cells in the adult zebrafish brain an organ that is characterized by pronounced zonation and high regenerative capacity. We combined single cell transcriptomics of dissected brain regions with massively parallel lineage tracing and in vivo RNA metabolic labeling to analyze regulation of neural stem cells in space and time. We detected a large diversity of neural stem cells with some subtypes being restricted to a single brain region while others were found globally across the brain. Global stem cell states are linked to neurogenic differentiation with different states being involved in proliferative and non proliferative differentiation. Our work reveals principles of adult stem cell organization and establishes a resource for functional manipulation of neural stem cell subtypes. Overall design: Single cell RNA seq of adult zebrafish brain performed using 10x Genomics Gene Expression protocols to explore transcriptomic diversity of adult cell types. The samples were prepared either from whole brain FACS sorted cells GFP positive cells from gfap:GFP line or a dissected single brain region as indicated in the sample title. CRISPR based lineage tracing was performed for a subset of samples to dissect developmental lineage relationships between cells. ScSLAM seq was performed in combination with Notch pathway inhibition to assess response of stem cells to perturbation.   pubmed:38365956   Lineage tracing rep1 rpl39 scars GSM7875191   source name:adult brain|tissue:adult brain|cell type:mixed tissue dissociation|genotype:Tg[ubi:zebrabow M]|geo loc name:missing|collection date:missing Lineage tracing rep1 rpl39 scars The transcriptome libraries were demultiplexed and mapped with CellRanger 6.1.1. In order to reduce batch effect the gene expression matrices were processed with the SoupX tool Young & Behjati 2020 for removing ambient RNA contamination. A subset of libraries was also mapped with Velocyto v0.17.17 La Manno et al 2018 to create count matrices for unspliced and spliced molecules. The targeted lineage tracing libraries were aligned using bwa mem3 v.0.7.12 to individual references of endogenous genes used in the lineage tracing experiment actb1 actb2 cfl1 cirbpb rpl39 and ube2e1. The scars were filtered with a custom pipeline to eliminate sequences that originate from PCR or sequencing errors. For custom code and further downstream analysis see: https://github.com/nimitic/radial glia. Assembly: GRCz11 Supplementary files format and content: Tab separated barcode files and matrix files along with the tab separated gene file supplementary file are the output of CellRanger mapping further processed by the SoupX package for ambient RNA removal. Supplementary files format and content: Loom files are the output of velocyto mapping distinguishing spliced and unspliced mRNAs. Supplementary files format and content: filtered scars.csv files contain the output of a custom scar filtering pipeline. adult brain   Adult zebrafish brain tissue was extracted optionally dissected to select a specific region of interest telencephalon diencephalon mesencephalon or rhombencephalon and then dissociated with a trypsin protocol. Briefly the samples were incubated in 750 µl of HBSS glucose solution and dissocated with 15 µl of 0.05% trypsin EDTA Gibco for 30 minutes at 37°C with intermittent mixing. The disociation was stopped by addition of 750 µl of a BSA EBSS HEPES solution. Further sample handling steps were carried out on ice and for centrifugation at 4°C. The sample was filtered through a 100 µm filter washed with cold HBSS and resuspended in 100 200 µl HBSS with 0.05% BSA then filtered through a 35 µm filter and loaded on the 10x Chromium Controller. For the scSLAM seq samples a papain dissociation protocol Worthington kit according to manufecturer's instructions was used followed by an adapted scSLAM seq protocol for labeling nascent transcripts see sample specific entry. The standard transcriptome libraries were constructed according to the manufacturer's protocol 10X Genomics. For targeted libraries of scars used for lineage tracing the cDNA obtained during the 10x protocol was used to set up individual PCR reactions for each target specifically amplifying the sgRNA target site. The libraries for targeted PCR were constructed with primers compatible with the 10X Genomics kit so they could be sequenced on the same platform.   tissue:adult brain|cell type:mixed tissue dissociation|genotype:Tg[ubi:zebrabow M] GSM7875191 GSM7875191: Lineage tracing rep1 rpl39 scars; Danio rerio; OTHER GSM7875191 r1 GSM7875191 1 Adult zebrafish brain tissue was extracted optionally dissected to select a specific region of interest telencephalon diencephalon mesencephalon or rhombencephalon and then dissociated with a trypsin protocol. Briefly the samples were incubated in 750 µl of HBSS glucose solution and dissocated with 15 µl of 0.05% trypsin EDTA Gibco for 30 minutes at 37°C with intermittent mixing. The disociation was stopped by addition of 750 µl of a BSA EBSS HEPES solution. Further sample handling steps were carried out on ice and for centrifugation at 4°C. The sample was filtered through a 100 µm filter washed with cold HBSS and resuspended in 100 200 µl HBSS with 0.05% BSA then filtered through a 35 µm filter and loaded on the 10x Chromium Controller. For the scSLAM seq samples a papain dissociation protocol Worthington kit according to manufecturer's instructions was used followed by an adapted scSLAM seq protocol for labeling nascent transcripts see sample specific entry. The standard transcriptome libraries were constructed according to the manufacturer's protocol 10X Genomics. For targeted libraries of scars used for lineage tracing the cDNA obtained during the 10x protocol was used to set up individual PCR reactions for each target specifically amplifying the sgRNA target site. The libraries for targeted PCR were constructed with primers compatible with the 10X Genomics kit so they could be sequenced on the same platform.   OTHER TRANSCRIPTOMIC other PAIRED ILLUMINA NextSeq 500   SRP469552   loader:fastq load.py lin1_rpl39_scar_R1.fastq.gz lin1_rpl39_scar_R2.fastq.gz fastq fastq 401898718.0 2276836.0 GSM7875191 r1                       SRX22323892 SRS19374422 SRA1743007 Junker Lab, Berlin Institute for Medical Systems Biology, Max-Delbrück-Center for Molecular Medicine Junker Lab, Berlin Institute for Medical Systems Biology, Max-Delbrück-Center for Molecular Medicine 2 0.00037 0.2642 0.00015 0.00086 0.99922 0.99026 0.23076 0.42455 28 120 T B sc-like readlen illumina nextseq unknown other unknown sc single_cell_droplet 10x   Germany 2023-10-31 Adult Adult Brain Nervous System
31943 31943 SRR28790250 SRX24354554 SRS21112322 SRP503769 PRJNA1104180 Double stranded RNA triggers a distinct integrated stress response in the early embryo [RNA seq Ribo seq] GSE265771 Other Double stranded RNA dsRNA is associated with virus infections and is present as by products during the transcription of synthetic mRNA which has been widely used in gene gain of function studies and serves as a core component in emerging mRNA based therapies1 4. The presence of dsRNA in host cells induces an integrated stress response that functions to prevent virus replication and infection5 6. Unlike differentiated cells undifferentiated cells adopt a distinct defense strategy against RNA virus infection7 but the mechanism is unclear. We show a previously unidentified response triggered by dsRNA in the early embryo. Although dsRNA causes a global protein translation inhibition in a PKR eIF2a independent manner and leads to developmental delay and cell necrosis it also strongly induces p53 activation which then upregulates Interferon Stimulated Genes independently of interferon ligands. Importantly we demonstrate that the burst of p53 signaling dose not result in cell death but functions as a protective mechanism against deleterious translation blockage by slowing down global protein degradation via ISGylation. Our work has identified a distinct dsRNA induced stress response in the embryo reflecting an ancient innate immune memory before the establishment of the IFN system. It also raises the provocative question as to the original protective role of p53 during evolution. Overall design: To characterize the distribution of ribosomes on mRNA we performed Ribosome profiling Ribo seq analysis with RNA seq on dsRNA injected embryos.       Zebrafish Riboseq high dose dsRNA2 GSM8228804   source name:whole embryo|tissue:whole embryo|developmental stage:50% epiboly stage|cell type:embryonic cell|genotype:wild type|treatment:dsRNA|geo loc name:missing|collection date:missing Zebrafish Riboseq high dose dsRNA2 The library underwent quality control assessment and was subjected to Illumina Novaseq 6000 sequencing. Bio informatics analysis of RIBOseq profilling was performed to analyze the library data. Assembly: GRCz10 Supplementary files format and content: Excel file includes raw counts for each Sample Library strategy: Ribo seq whole embryo   Both control group and dsRNA injected embryos at xxx hpf were thoroughly lysed on ice using lysis buffer.The lysate was then centrifuged at 12000g at 4°C for 10 minutes to remove cellular components including undigested material such as cell nuclei.Then RNaseI was added to the supernatant and incubated at 25°C for 30 minutes to digest RNA while the RNA fragments protected by ribosomes were preserved. Subsequently an RNA inhibitor was added to terminate the digestion reaction. The lysate was subjected to sucrose density gradient centrifugation and fractions containing single peaks at 260nm wavelength around the 80S monosome region were collected. The collected mixture was then thoroughly lysed using Trizol for RNA extraction. RNA fragments within the size range of 26 34nt was separated using polyacrylamide gel electrophoresis without xxx and the corresponding bands were collected. The recovered bands were subjected to ligation reaction where a preadenylylated and 3’ blocked linker 5’ rApp CTGTAGGCACCATCAAT NH2 3’ was attached to the three prime end of the RNA. The ligated products were then recovered by performing polyacrylamide gel electrophoresis. Subsequently the recovered RNA fragments were reverse transcribed using a reverse primer to obtain extended cDNA molecules. The purified cDNA was subjected to circularization by using CircLigase Epicentre CL4111K resulting in the formation of circular cDNA molecules. To remove residual rRNA components in the cDNA a method involving hybridization with complementary primers specific to rRNA and subsequent heat denaturation was employed. The remaining circularized cDNA was then amplified through PCR and barcode sequences were incorporated.   tissue:whole embryo|developmental stage:50% epiboly stage|cell type:embryonic cell|genotype:wild type|treatment:dsRNA GSM8228804 GSM8228804: Zebrafish Riboseq high dose dsRNA2; Danio rerio; OTHER GSM8228804 r1 GSM8228804 1 Both control group and dsRNA injected embryos at xxx hpf were thoroughly lysed on ice using lysis buffer.The lysate was then centrifuged at 12000g at 4°C for 10 minutes to remove cellular components including undigested material such as cell nuclei.Then RNaseI was added to the supernatant and incubated at 25°C for 30 minutes to digest RNA while the RNA fragments protected by ribosomes were preserved. Subsequently an RNA inhibitor was added to terminate the digestion reaction. The lysate was subjected to sucrose density gradient centrifugation and fractions containing single peaks at 260nm wavelength around the 80S monosome region were collected. The collected mixture was then thoroughly lysed using Trizol for RNA extraction. RNA fragments within the size range of 26 34nt was separated using polyacrylamide gel electrophoresis without xxx and the corresponding bands were collected. The recovered bands were subjected to ligation reaction where a preadenylylated and three prime blocked linker five prime rApp CTGTAGGCACCATCAAT NH2 three prime was attached to the three prime end of the RNA. The ligated products were then recovered by performing polyacrylamide gel electrophoresis. Subsequently the recovered RNA fragments were reverse transcribed using a reverse primer to obtain extended cDNA molecules. The purified cDNA was subjected to circularization by using CircLigase Epicentre CL4111K resulting in the formation of circular cDNA molecules. To remove residual rRNA components in the cDNA a method involving hybridization with complementary primers specific to rRNA and subsequent heat denaturation was employed. The remaining circularized cDNA was then amplified through PCR and barcode sequences were incorporated.   OTHER TRANSCRIPTOMIC other PAIRED ILLUMINA Illumina NovaSeq 6000   SRP503769     dsR_2.R1.raw.fastq.gz dsR_2.R2.raw.fastq.gz fastq fastq 15877367026.0 52574063.0 GSM8228804 r1 0:151 1:151 A:3017944891;C:2902425937;G:7224440180;T:2731881397;N:674621 151 151     3017944891 2902425937 7224440180 2731881397 674621 SRX24354554 SRS21112322 SRA1852128 ShanDong University ShanDong University                       T T mates < 9% mapping rate illumina novaseq_era 3prime random_priming unknown bulk unknown unknown   China 2024-04-24 Gastrula Embryo Whole Organism All anatomical structures
31944 31944 SRR28790251 SRX24354553 SRS21112321 SRP503769 PRJNA1104180 Double stranded RNA triggers a distinct integrated stress response in the early embryo [RNA seq Ribo seq] GSE265771 Other Double stranded RNA dsRNA is associated with virus infections and is present as by products during the transcription of synthetic mRNA which has been widely used in gene gain of function studies and serves as a core component in emerging mRNA based therapies1 4. The presence of dsRNA in host cells induces an integrated stress response that functions to prevent virus replication and infection5 6. Unlike differentiated cells undifferentiated cells adopt a distinct defense strategy against RNA virus infection7 but the mechanism is unclear. We show a previously unidentified response triggered by dsRNA in the early embryo. Although dsRNA causes a global protein translation inhibition in a PKR eIF2a independent manner and leads to developmental delay and cell necrosis it also strongly induces p53 activation which then upregulates Interferon Stimulated Genes independently of interferon ligands. Importantly we demonstrate that the burst of p53 signaling dose not result in cell death but functions as a protective mechanism against deleterious translation blockage by slowing down global protein degradation via ISGylation. Our work has identified a distinct dsRNA induced stress response in the embryo reflecting an ancient innate immune memory before the establishment of the IFN system. It also raises the provocative question as to the original protective role of p53 during evolution. Overall design: To characterize the distribution of ribosomes on mRNA we performed Ribosome profiling Ribo seq analysis with RNA seq on dsRNA injected embryos.       Zebrafish Riboseq high dose dsRNA1 GSM8228803   source name:whole embryo|tissue:whole embryo|developmental stage:50% epiboly stage|cell type:embryonic cell|genotype:wild type|treatment:dsRNA|geo loc name:missing|collection date:missing Zebrafish Riboseq high dose dsRNA1 The library underwent quality control assessment and was subjected to Illumina Novaseq 6000 sequencing. Bio informatics analysis of RIBOseq profilling was performed to analyze the library data. Assembly: GRCz10 Supplementary files format and content: Excel file includes raw counts for each Sample Library strategy: Ribo seq whole embryo   Both control group and dsRNA injected embryos at xxx hpf were thoroughly lysed on ice using lysis buffer.The lysate was then centrifuged at 12000g at 4°C for 10 minutes to remove cellular components including undigested material such as cell nuclei.Then RNaseI was added to the supernatant and incubated at 25°C for 30 minutes to digest RNA while the RNA fragments protected by ribosomes were preserved. Subsequently an RNA inhibitor was added to terminate the digestion reaction. The lysate was subjected to sucrose density gradient centrifugation and fractions containing single peaks at 260nm wavelength around the 80S monosome region were collected. The collected mixture was then thoroughly lysed using Trizol for RNA extraction. RNA fragments within the size range of 26 34nt was separated using polyacrylamide gel electrophoresis without xxx and the corresponding bands were collected. The recovered bands were subjected to ligation reaction where a preadenylylated and 3’ blocked linker 5’ rApp CTGTAGGCACCATCAAT NH2 3’ was attached to the three prime end of the RNA. The ligated products were then recovered by performing polyacrylamide gel electrophoresis. Subsequently the recovered RNA fragments were reverse transcribed using a reverse primer to obtain extended cDNA molecules. The purified cDNA was subjected to circularization by using CircLigase Epicentre CL4111K resulting in the formation of circular cDNA molecules. To remove residual rRNA components in the cDNA a method involving hybridization with complementary primers specific to rRNA and subsequent heat denaturation was employed. The remaining circularized cDNA was then amplified through PCR and barcode sequences were incorporated.   tissue:whole embryo|developmental stage:50% epiboly stage|cell type:embryonic cell|genotype:wild type|treatment:dsRNA GSM8228803 GSM8228803: Zebrafish Riboseq high dose dsRNA1; Danio rerio; OTHER GSM8228803 r1 GSM8228803 1 Both control group and dsRNA injected embryos at xxx hpf were thoroughly lysed on ice using lysis buffer.The lysate was then centrifuged at 12000g at 4°C for 10 minutes to remove cellular components including undigested material such as cell nuclei.Then RNaseI was added to the supernatant and incubated at 25°C for 30 minutes to digest RNA while the RNA fragments protected by ribosomes were preserved. Subsequently an RNA inhibitor was added to terminate the digestion reaction. The lysate was subjected to sucrose density gradient centrifugation and fractions containing single peaks at 260nm wavelength around the 80S monosome region were collected. The collected mixture was then thoroughly lysed using Trizol for RNA extraction. RNA fragments within the size range of 26 34nt was separated using polyacrylamide gel electrophoresis without xxx and the corresponding bands were collected. The recovered bands were subjected to ligation reaction where a preadenylylated and three prime blocked linker five prime rApp CTGTAGGCACCATCAAT NH2 three prime was attached to the three prime end of the RNA. The ligated products were then recovered by performing polyacrylamide gel electrophoresis. Subsequently the recovered RNA fragments were reverse transcribed using a reverse primer to obtain extended cDNA molecules. The purified cDNA was subjected to circularization by using CircLigase Epicentre CL4111K resulting in the formation of circular cDNA molecules. To remove residual rRNA components in the cDNA a method involving hybridization with complementary primers specific to rRNA and subsequent heat denaturation was employed. The remaining circularized cDNA was then amplified through PCR and barcode sequences were incorporated.   OTHER TRANSCRIPTOMIC other PAIRED ILLUMINA Illumina NovaSeq 6000   SRP503769     dsR_1.R1.raw.fastq.gz dsR_1.R2.raw.fastq.gz fastq fastq 13027928774.0 43138837.0 GSM8228803 r1 0:151 1:151 A:2538099201;C:2400262316;G:5852047273;T:2236961902;N:558082 151 151     2538099201 2400262316 5852047273 2236961902 558082 SRX24354553 SRS21112321 SRA1852128 ShanDong University ShanDong University                       T T mates < 9% mapping rate illumina novaseq_era 3prime random_priming unknown bulk unknown unknown   China 2024-04-24 Gastrula Embryo Whole Organism All anatomical structures
31945 31945 SRR28790252 SRX24354552 SRS21112320 SRP503769 PRJNA1104180 Double stranded RNA triggers a distinct integrated stress response in the early embryo [RNA seq Ribo seq] GSE265771 Other Double stranded RNA dsRNA is associated with virus infections and is present as by products during the transcription of synthetic mRNA which has been widely used in gene gain of function studies and serves as a core component in emerging mRNA based therapies1 4. The presence of dsRNA in host cells induces an integrated stress response that functions to prevent virus replication and infection5 6. Unlike differentiated cells undifferentiated cells adopt a distinct defense strategy against RNA virus infection7 but the mechanism is unclear. We show a previously unidentified response triggered by dsRNA in the early embryo. Although dsRNA causes a global protein translation inhibition in a PKR eIF2a independent manner and leads to developmental delay and cell necrosis it also strongly induces p53 activation which then upregulates Interferon Stimulated Genes independently of interferon ligands. Importantly we demonstrate that the burst of p53 signaling dose not result in cell death but functions as a protective mechanism against deleterious translation blockage by slowing down global protein degradation via ISGylation. Our work has identified a distinct dsRNA induced stress response in the embryo reflecting an ancient innate immune memory before the establishment of the IFN system. It also raises the provocative question as to the original protective role of p53 during evolution. Overall design: To characterize the distribution of ribosomes on mRNA we performed Ribosome profiling Ribo seq analysis with RNA seq on dsRNA injected embryos.       Zebrafish Riboseq high dose uninj2 GSM8228802   source name:whole embryo|tissue:whole embryo|developmental stage:50% epiboly stage|cell type:embryonic cell|genotype:wild type|treatment:uninjected|geo loc name:missing|collection date:missing Zebrafish Riboseq high dose uninj2 The library underwent quality control assessment and was subjected to Illumina Novaseq 6000 sequencing. Bio informatics analysis of RIBOseq profilling was performed to analyze the library data. Assembly: GRCz10 Supplementary files format and content: Excel file includes raw counts for each Sample Library strategy: Ribo seq whole embryo   Both control group and dsRNA injected embryos at xxx hpf were thoroughly lysed on ice using lysis buffer.The lysate was then centrifuged at 12000g at 4°C for 10 minutes to remove cellular components including undigested material such as cell nuclei.Then RNaseI was added to the supernatant and incubated at 25°C for 30 minutes to digest RNA while the RNA fragments protected by ribosomes were preserved. Subsequently an RNA inhibitor was added to terminate the digestion reaction. The lysate was subjected to sucrose density gradient centrifugation and fractions containing single peaks at 260nm wavelength around the 80S monosome region were collected. The collected mixture was then thoroughly lysed using Trizol for RNA extraction. RNA fragments within the size range of 26 34nt was separated using polyacrylamide gel electrophoresis without xxx and the corresponding bands were collected. The recovered bands were subjected to ligation reaction where a preadenylylated and 3’ blocked linker 5’ rApp CTGTAGGCACCATCAAT NH2 3’ was attached to the three prime end of the RNA. The ligated products were then recovered by performing polyacrylamide gel electrophoresis. Subsequently the recovered RNA fragments were reverse transcribed using a reverse primer to obtain extended cDNA molecules. The purified cDNA was subjected to circularization by using CircLigase Epicentre CL4111K resulting in the formation of circular cDNA molecules. To remove residual rRNA components in the cDNA a method involving hybridization with complementary primers specific to rRNA and subsequent heat denaturation was employed. The remaining circularized cDNA was then amplified through PCR and barcode sequences were incorporated.   tissue:whole embryo|developmental stage:50% epiboly stage|cell type:embryonic cell|genotype:wild type|treatment:uninjected GSM8228802 GSM8228802: Zebrafish Riboseq high dose uninj2; Danio rerio; OTHER GSM8228802 r1 GSM8228802 1 Both control group and dsRNA injected embryos at xxx hpf were thoroughly lysed on ice using lysis buffer.The lysate was then centrifuged at 12000g at 4°C for 10 minutes to remove cellular components including undigested material such as cell nuclei.Then RNaseI was added to the supernatant and incubated at 25°C for 30 minutes to digest RNA while the RNA fragments protected by ribosomes were preserved. Subsequently an RNA inhibitor was added to terminate the digestion reaction. The lysate was subjected to sucrose density gradient centrifugation and fractions containing single peaks at 260nm wavelength around the 80S monosome region were collected. The collected mixture was then thoroughly lysed using Trizol for RNA extraction. RNA fragments within the size range of 26 34nt was separated using polyacrylamide gel electrophoresis without xxx and the corresponding bands were collected. The recovered bands were subjected to ligation reaction where a preadenylylated and three prime blocked linker five prime rApp CTGTAGGCACCATCAAT NH2 three prime was attached to the three prime end of the RNA. The ligated products were then recovered by performing polyacrylamide gel electrophoresis. Subsequently the recovered RNA fragments were reverse transcribed using a reverse primer to obtain extended cDNA molecules. The purified cDNA was subjected to circularization by using CircLigase Epicentre CL4111K resulting in the formation of circular cDNA molecules. To remove residual rRNA components in the cDNA a method involving hybridization with complementary primers specific to rRNA and subsequent heat denaturation was employed. The remaining circularized cDNA was then amplified through PCR and barcode sequences were incorporated.   OTHER TRANSCRIPTOMIC other PAIRED ILLUMINA Illumina NovaSeq 6000   SRP503769     un_2.R1.raw.fastq.gz un_2.R2.raw.fastq.gz fastq fastq 13637060056.0 45155828.0 GSM8228802 r1 0:151 1:151 A:2596015831;C:2388366938;G:6201010197;T:2451085273;N:581817 151 151     2596015831 2388366938 6201010197 2451085273 581817 SRX24354552 SRS21112320 SRA1852128 ShanDong University ShanDong University                       T T mates < 9% mapping rate illumina novaseq_era 3prime random_priming unknown bulk unknown unknown   China 2024-04-24 Gastrula Embryo Whole Organism All anatomical structures
31946 31946 SRR28790253 SRX24354551 SRS21112319 SRP503769 PRJNA1104180 Double stranded RNA triggers a distinct integrated stress response in the early embryo [RNA seq Ribo seq] GSE265771 Other Double stranded RNA dsRNA is associated with virus infections and is present as by products during the transcription of synthetic mRNA which has been widely used in gene gain of function studies and serves as a core component in emerging mRNA based therapies1 4. The presence of dsRNA in host cells induces an integrated stress response that functions to prevent virus replication and infection5 6. Unlike differentiated cells undifferentiated cells adopt a distinct defense strategy against RNA virus infection7 but the mechanism is unclear. We show a previously unidentified response triggered by dsRNA in the early embryo. Although dsRNA causes a global protein translation inhibition in a PKR eIF2a independent manner and leads to developmental delay and cell necrosis it also strongly induces p53 activation which then upregulates Interferon Stimulated Genes independently of interferon ligands. Importantly we demonstrate that the burst of p53 signaling dose not result in cell death but functions as a protective mechanism against deleterious translation blockage by slowing down global protein degradation via ISGylation. Our work has identified a distinct dsRNA induced stress response in the embryo reflecting an ancient innate immune memory before the establishment of the IFN system. It also raises the provocative question as to the original protective role of p53 during evolution. Overall design: To characterize the distribution of ribosomes on mRNA we performed Ribosome profiling Ribo seq analysis with RNA seq on dsRNA injected embryos.       Zebrafish Riboseq high dose uninj1 GSM8228801   source name:whole embryo|tissue:whole embryo|developmental stage:50% epiboly stage|cell type:embryonic cell|genotype:wild type|treatment:uninjected|geo loc name:missing|collection date:missing Zebrafish Riboseq high dose uninj1 The library underwent quality control assessment and was subjected to Illumina Novaseq 6000 sequencing. Bio informatics analysis of RIBOseq profilling was performed to analyze the library data. Assembly: GRCz10 Supplementary files format and content: Excel file includes raw counts for each Sample Library strategy: Ribo seq whole embryo   Both control group and dsRNA injected embryos at xxx hpf were thoroughly lysed on ice using lysis buffer.The lysate was then centrifuged at 12000g at 4°C for 10 minutes to remove cellular components including undigested material such as cell nuclei.Then RNaseI was added to the supernatant and incubated at 25°C for 30 minutes to digest RNA while the RNA fragments protected by ribosomes were preserved. Subsequently an RNA inhibitor was added to terminate the digestion reaction. The lysate was subjected to sucrose density gradient centrifugation and fractions containing single peaks at 260nm wavelength around the 80S monosome region were collected. The collected mixture was then thoroughly lysed using Trizol for RNA extraction. RNA fragments within the size range of 26 34nt was separated using polyacrylamide gel electrophoresis without xxx and the corresponding bands were collected. The recovered bands were subjected to ligation reaction where a preadenylylated and 3’ blocked linker 5’ rApp CTGTAGGCACCATCAAT NH2 3’ was attached to the three prime end of the RNA. The ligated products were then recovered by performing polyacrylamide gel electrophoresis. Subsequently the recovered RNA fragments were reverse transcribed using a reverse primer to obtain extended cDNA molecules. The purified cDNA was subjected to circularization by using CircLigase Epicentre CL4111K resulting in the formation of circular cDNA molecules. To remove residual rRNA components in the cDNA a method involving hybridization with complementary primers specific to rRNA and subsequent heat denaturation was employed. The remaining circularized cDNA was then amplified through PCR and barcode sequences were incorporated.   tissue:whole embryo|developmental stage:50% epiboly stage|cell type:embryonic cell|genotype:wild type|treatment:uninjected GSM8228801 GSM8228801: Zebrafish Riboseq high dose uninj1; Danio rerio; OTHER GSM8228801 r1 GSM8228801 1 Both control group and dsRNA injected embryos at xxx hpf were thoroughly lysed on ice using lysis buffer.The lysate was then centrifuged at 12000g at 4°C for 10 minutes to remove cellular components including undigested material such as cell nuclei.Then RNaseI was added to the supernatant and incubated at 25°C for 30 minutes to digest RNA while the RNA fragments protected by ribosomes were preserved. Subsequently an RNA inhibitor was added to terminate the digestion reaction. The lysate was subjected to sucrose density gradient centrifugation and fractions containing single peaks at 260nm wavelength around the 80S monosome region were collected. The collected mixture was then thoroughly lysed using Trizol for RNA extraction. RNA fragments within the size range of 26 34nt was separated using polyacrylamide gel electrophoresis without xxx and the corresponding bands were collected. The recovered bands were subjected to ligation reaction where a preadenylylated and three prime blocked linker five prime rApp CTGTAGGCACCATCAAT NH2 three prime was attached to the three prime end of the RNA. The ligated products were then recovered by performing polyacrylamide gel electrophoresis. Subsequently the recovered RNA fragments were reverse transcribed using a reverse primer to obtain extended cDNA molecules. The purified cDNA was subjected to circularization by using CircLigase Epicentre CL4111K resulting in the formation of circular cDNA molecules. To remove residual rRNA components in the cDNA a method involving hybridization with complementary primers specific to rRNA and subsequent heat denaturation was employed. The remaining circularized cDNA was then amplified through PCR and barcode sequences were incorporated.   OTHER TRANSCRIPTOMIC other PAIRED ILLUMINA Illumina NovaSeq 6000   SRP503769     un_1.R1.raw.fastq.gz un_1.R2.raw.fastq.gz fastq fastq 13499333560.0 44699780.0 GSM8228801 r1 0:151 1:151 A:2587725963;C:2439789499;G:6080777505;T:2390460459;N:580134 151 151     2587725963 2439789499 6080777505 2390460459 580134 SRX24354551 SRS21112319 SRA1852128 ShanDong University ShanDong University                       T T mates < 9% mapping rate illumina novaseq_era 3prime random_priming unknown bulk unknown unknown   China 2024-04-24 Gastrula Embryo Whole Organism All anatomical structures
31947 31947 SRR28790254 SRX24354550 SRS21112318 SRP503769 PRJNA1104180 Double stranded RNA triggers a distinct integrated stress response in the early embryo [RNA seq Ribo seq] GSE265771 Other Double stranded RNA dsRNA is associated with virus infections and is present as by products during the transcription of synthetic mRNA which has been widely used in gene gain of function studies and serves as a core component in emerging mRNA based therapies1 4. The presence of dsRNA in host cells induces an integrated stress response that functions to prevent virus replication and infection5 6. Unlike differentiated cells undifferentiated cells adopt a distinct defense strategy against RNA virus infection7 but the mechanism is unclear. We show a previously unidentified response triggered by dsRNA in the early embryo. Although dsRNA causes a global protein translation inhibition in a PKR eIF2a independent manner and leads to developmental delay and cell necrosis it also strongly induces p53 activation which then upregulates Interferon Stimulated Genes independently of interferon ligands. Importantly we demonstrate that the burst of p53 signaling dose not result in cell death but functions as a protective mechanism against deleterious translation blockage by slowing down global protein degradation via ISGylation. Our work has identified a distinct dsRNA induced stress response in the embryo reflecting an ancient innate immune memory before the establishment of the IFN system. It also raises the provocative question as to the original protective role of p53 during evolution. Overall design: To characterize the distribution of ribosomes on mRNA we performed Ribosome profiling Ribo seq analysis with RNA seq on dsRNA injected embryos.       Zebrafish Riboseq low dose dsRNA2 GSM8228800   source name:whole embryo|tissue:whole embryo|developmental stage:50% epiboly stage|cell type:embryonic cell|genotype:wild type|treatment:dsRNA|geo loc name:missing|collection date:missing Zebrafish Riboseq low dose dsRNA2 The library underwent quality control assessment and was subjected to Illumina Novaseq 6000 sequencing. Bio informatics analysis of RIBOseq profilling was performed to analyze the library data. Assembly: GRCz10 Supplementary files format and content: Excel file includes raw counts for each Sample Library strategy: Ribo seq whole embryo   Both control group and dsRNA injected embryos at xxx hpf were thoroughly lysed on ice using lysis buffer.The lysate was then centrifuged at 12000g at 4°C for 10 minutes to remove cellular components including undigested material such as cell nuclei.Then RNaseI was added to the supernatant and incubated at 25°C for 30 minutes to digest RNA while the RNA fragments protected by ribosomes were preserved. Subsequently an RNA inhibitor was added to terminate the digestion reaction. The lysate was subjected to sucrose density gradient centrifugation and fractions containing single peaks at 260nm wavelength around the 80S monosome region were collected. The collected mixture was then thoroughly lysed using Trizol for RNA extraction. RNA fragments within the size range of 26 34nt was separated using polyacrylamide gel electrophoresis without xxx and the corresponding bands were collected. The recovered bands were subjected to ligation reaction where a preadenylylated and 3’ blocked linker 5’ rApp CTGTAGGCACCATCAAT NH2 3’ was attached to the three prime end of the RNA. The ligated products were then recovered by performing polyacrylamide gel electrophoresis. Subsequently the recovered RNA fragments were reverse transcribed using a reverse primer to obtain extended cDNA molecules. The purified cDNA was subjected to circularization by using CircLigase Epicentre CL4111K resulting in the formation of circular cDNA molecules. To remove residual rRNA components in the cDNA a method involving hybridization with complementary primers specific to rRNA and subsequent heat denaturation was employed. The remaining circularized cDNA was then amplified through PCR and barcode sequences were incorporated.   tissue:whole embryo|developmental stage:50% epiboly stage|cell type:embryonic cell|genotype:wild type|treatment:dsRNA GSM8228800 GSM8228800: Zebrafish Riboseq low dose dsRNA2; Danio rerio; OTHER GSM8228800 r1 GSM8228800 1 Both control group and dsRNA injected embryos at xxx hpf were thoroughly lysed on ice using lysis buffer.The lysate was then centrifuged at 12000g at 4°C for 10 minutes to remove cellular components including undigested material such as cell nuclei.Then RNaseI was added to the supernatant and incubated at 25°C for 30 minutes to digest RNA while the RNA fragments protected by ribosomes were preserved. Subsequently an RNA inhibitor was added to terminate the digestion reaction. The lysate was subjected to sucrose density gradient centrifugation and fractions containing single peaks at 260nm wavelength around the 80S monosome region were collected. The collected mixture was then thoroughly lysed using Trizol for RNA extraction. RNA fragments within the size range of 26 34nt was separated using polyacrylamide gel electrophoresis without xxx and the corresponding bands were collected. The recovered bands were subjected to ligation reaction where a preadenylylated and three prime blocked linker five prime rApp CTGTAGGCACCATCAAT NH2 three prime was attached to the three prime end of the RNA. The ligated products were then recovered by performing polyacrylamide gel electrophoresis. Subsequently the recovered RNA fragments were reverse transcribed using a reverse primer to obtain extended cDNA molecules. The purified cDNA was subjected to circularization by using CircLigase Epicentre CL4111K resulting in the formation of circular cDNA molecules. To remove residual rRNA components in the cDNA a method involving hybridization with complementary primers specific to rRNA and subsequent heat denaturation was employed. The remaining circularized cDNA was then amplified through PCR and barcode sequences were incorporated.   OTHER TRANSCRIPTOMIC other PAIRED ILLUMINA Illumina NovaSeq 6000   SRP503769     dsR_2.raw.1.fastq.gz dsR_2.raw.2.fastq.gz fastq fastq 13335544766.0 44157433.0 GSM8228800 r1 0:151 1:151 A:2383528724;C:2262795235;G:6593680813;T:2094730217;N:809777 151 151     2383528724 2262795235 6593680813 2094730217 809777 SRX24354550 SRS21112318 SRA1852128 ShanDong University ShanDong University                       T T mates < 9% mapping rate illumina novaseq_era 3prime random_priming unknown bulk unknown unknown   China 2024-04-24 Gastrula Embryo Whole Organism All anatomical structures
31948 31948 SRR28790255 SRX24354549 SRS21112317 SRP503769 PRJNA1104180 Double stranded RNA triggers a distinct integrated stress response in the early embryo [RNA seq Ribo seq] GSE265771 Other Double stranded RNA dsRNA is associated with virus infections and is present as by products during the transcription of synthetic mRNA which has been widely used in gene gain of function studies and serves as a core component in emerging mRNA based therapies1 4. The presence of dsRNA in host cells induces an integrated stress response that functions to prevent virus replication and infection5 6. Unlike differentiated cells undifferentiated cells adopt a distinct defense strategy against RNA virus infection7 but the mechanism is unclear. We show a previously unidentified response triggered by dsRNA in the early embryo. Although dsRNA causes a global protein translation inhibition in a PKR eIF2a independent manner and leads to developmental delay and cell necrosis it also strongly induces p53 activation which then upregulates Interferon Stimulated Genes independently of interferon ligands. Importantly we demonstrate that the burst of p53 signaling dose not result in cell death but functions as a protective mechanism against deleterious translation blockage by slowing down global protein degradation via ISGylation. Our work has identified a distinct dsRNA induced stress response in the embryo reflecting an ancient innate immune memory before the establishment of the IFN system. It also raises the provocative question as to the original protective role of p53 during evolution. Overall design: To characterize the distribution of ribosomes on mRNA we performed Ribosome profiling Ribo seq analysis with RNA seq on dsRNA injected embryos.       Zebrafish Riboseq low dose dsRNA1 GSM8228799   source name:whole embryo|tissue:whole embryo|developmental stage:50% epiboly stage|cell type:embryonic cell|genotype:wild type|treatment:dsRNA|geo loc name:missing|collection date:missing Zebrafish Riboseq low dose dsRNA1 The library underwent quality control assessment and was subjected to Illumina Novaseq 6000 sequencing. Bio informatics analysis of RIBOseq profilling was performed to analyze the library data. Assembly: GRCz10 Supplementary files format and content: Excel file includes raw counts for each Sample Library strategy: Ribo seq whole embryo   Both control group and dsRNA injected embryos at xxx hpf were thoroughly lysed on ice using lysis buffer.The lysate was then centrifuged at 12000g at 4°C for 10 minutes to remove cellular components including undigested material such as cell nuclei.Then RNaseI was added to the supernatant and incubated at 25°C for 30 minutes to digest RNA while the RNA fragments protected by ribosomes were preserved. Subsequently an RNA inhibitor was added to terminate the digestion reaction. The lysate was subjected to sucrose density gradient centrifugation and fractions containing single peaks at 260nm wavelength around the 80S monosome region were collected. The collected mixture was then thoroughly lysed using Trizol for RNA extraction. RNA fragments within the size range of 26 34nt was separated using polyacrylamide gel electrophoresis without xxx and the corresponding bands were collected. The recovered bands were subjected to ligation reaction where a preadenylylated and 3’ blocked linker 5’ rApp CTGTAGGCACCATCAAT NH2 3’ was attached to the three prime end of the RNA. The ligated products were then recovered by performing polyacrylamide gel electrophoresis. Subsequently the recovered RNA fragments were reverse transcribed using a reverse primer to obtain extended cDNA molecules. The purified cDNA was subjected to circularization by using CircLigase Epicentre CL4111K resulting in the formation of circular cDNA molecules. To remove residual rRNA components in the cDNA a method involving hybridization with complementary primers specific to rRNA and subsequent heat denaturation was employed. The remaining circularized cDNA was then amplified through PCR and barcode sequences were incorporated.   tissue:whole embryo|developmental stage:50% epiboly stage|cell type:embryonic cell|genotype:wild type|treatment:dsRNA GSM8228799 GSM8228799: Zebrafish Riboseq low dose dsRNA1; Danio rerio; OTHER GSM8228799 r1 GSM8228799 1 Both control group and dsRNA injected embryos at xxx hpf were thoroughly lysed on ice using lysis buffer.The lysate was then centrifuged at 12000g at 4°C for 10 minutes to remove cellular components including undigested material such as cell nuclei.Then RNaseI was added to the supernatant and incubated at 25°C for 30 minutes to digest RNA while the RNA fragments protected by ribosomes were preserved. Subsequently an RNA inhibitor was added to terminate the digestion reaction. The lysate was subjected to sucrose density gradient centrifugation and fractions containing single peaks at 260nm wavelength around the 80S monosome region were collected. The collected mixture was then thoroughly lysed using Trizol for RNA extraction. RNA fragments within the size range of 26 34nt was separated using polyacrylamide gel electrophoresis without xxx and the corresponding bands were collected. The recovered bands were subjected to ligation reaction where a preadenylylated and three prime blocked linker five prime rApp CTGTAGGCACCATCAAT NH2 three prime was attached to the three prime end of the RNA. The ligated products were then recovered by performing polyacrylamide gel electrophoresis. Subsequently the recovered RNA fragments were reverse transcribed using a reverse primer to obtain extended cDNA molecules. The purified cDNA was subjected to circularization by using CircLigase Epicentre CL4111K resulting in the formation of circular cDNA molecules. To remove residual rRNA components in the cDNA a method involving hybridization with complementary primers specific to rRNA and subsequent heat denaturation was employed. The remaining circularized cDNA was then amplified through PCR and barcode sequences were incorporated.   OTHER TRANSCRIPTOMIC other PAIRED ILLUMINA Illumina NovaSeq 6000   SRP503769     dsR_1.raw.1.fastq.gz dsR_1.raw.2.fastq.gz fastq fastq 12530433302.0 41491501.0 GSM8228799 r1 0:151 1:151 A:2300207705;C:2164355626;G:6050556625;T:2014558360;N:754986 151 151     2300207705 2164355626 6050556625 2014558360 754986 SRX24354549 SRS21112317 SRA1852128 ShanDong University ShanDong University                       T T mates < 9% mapping rate illumina novaseq_era 3prime random_priming unknown bulk unknown unknown   China 2024-04-24 Gastrula Embryo Whole Organism All anatomical structures
31949 31949 SRR28790256 SRX24354548 SRS21112316 SRP503769 PRJNA1104180 Double stranded RNA triggers a distinct integrated stress response in the early embryo [RNA seq Ribo seq] GSE265771 Other Double stranded RNA dsRNA is associated with virus infections and is present as by products during the transcription of synthetic mRNA which has been widely used in gene gain of function studies and serves as a core component in emerging mRNA based therapies1 4. The presence of dsRNA in host cells induces an integrated stress response that functions to prevent virus replication and infection5 6. Unlike differentiated cells undifferentiated cells adopt a distinct defense strategy against RNA virus infection7 but the mechanism is unclear. We show a previously unidentified response triggered by dsRNA in the early embryo. Although dsRNA causes a global protein translation inhibition in a PKR eIF2a independent manner and leads to developmental delay and cell necrosis it also strongly induces p53 activation which then upregulates Interferon Stimulated Genes independently of interferon ligands. Importantly we demonstrate that the burst of p53 signaling dose not result in cell death but functions as a protective mechanism against deleterious translation blockage by slowing down global protein degradation via ISGylation. Our work has identified a distinct dsRNA induced stress response in the embryo reflecting an ancient innate immune memory before the establishment of the IFN system. It also raises the provocative question as to the original protective role of p53 during evolution. Overall design: To characterize the distribution of ribosomes on mRNA we performed Ribosome profiling Ribo seq analysis with RNA seq on dsRNA injected embryos.       Zebrafish Riboseq low dose uninj2 GSM8228798   source name:whole embryo|tissue:whole embryo|developmental stage:50% epiboly stage|cell type:embryonic cell|genotype:wild type|treatment:uninjected|geo loc name:missing|collection date:missing Zebrafish Riboseq low dose uninj2 The library underwent quality control assessment and was subjected to Illumina Novaseq 6000 sequencing. Bio informatics analysis of RIBOseq profilling was performed to analyze the library data. Assembly: GRCz10 Supplementary files format and content: Excel file includes raw counts for each Sample Library strategy: Ribo seq whole embryo   Both control group and dsRNA injected embryos at xxx hpf were thoroughly lysed on ice using lysis buffer.The lysate was then centrifuged at 12000g at 4°C for 10 minutes to remove cellular components including undigested material such as cell nuclei.Then RNaseI was added to the supernatant and incubated at 25°C for 30 minutes to digest RNA while the RNA fragments protected by ribosomes were preserved. Subsequently an RNA inhibitor was added to terminate the digestion reaction. The lysate was subjected to sucrose density gradient centrifugation and fractions containing single peaks at 260nm wavelength around the 80S monosome region were collected. The collected mixture was then thoroughly lysed using Trizol for RNA extraction. RNA fragments within the size range of 26 34nt was separated using polyacrylamide gel electrophoresis without xxx and the corresponding bands were collected. The recovered bands were subjected to ligation reaction where a preadenylylated and 3’ blocked linker 5’ rApp CTGTAGGCACCATCAAT NH2 3’ was attached to the three prime end of the RNA. The ligated products were then recovered by performing polyacrylamide gel electrophoresis. Subsequently the recovered RNA fragments were reverse transcribed using a reverse primer to obtain extended cDNA molecules. The purified cDNA was subjected to circularization by using CircLigase Epicentre CL4111K resulting in the formation of circular cDNA molecules. To remove residual rRNA components in the cDNA a method involving hybridization with complementary primers specific to rRNA and subsequent heat denaturation was employed. The remaining circularized cDNA was then amplified through PCR and barcode sequences were incorporated.   tissue:whole embryo|developmental stage:50% epiboly stage|cell type:embryonic cell|genotype:wild type|treatment:uninjected GSM8228798 GSM8228798: Zebrafish Riboseq low dose uninj2; Danio rerio; OTHER GSM8228798 r1 GSM8228798 1 Both control group and dsRNA injected embryos at xxx hpf were thoroughly lysed on ice using lysis buffer.The lysate was then centrifuged at 12000g at 4°C for 10 minutes to remove cellular components including undigested material such as cell nuclei.Then RNaseI was added to the supernatant and incubated at 25°C for 30 minutes to digest RNA while the RNA fragments protected by ribosomes were preserved. Subsequently an RNA inhibitor was added to terminate the digestion reaction. The lysate was subjected to sucrose density gradient centrifugation and fractions containing single peaks at 260nm wavelength around the 80S monosome region were collected. The collected mixture was then thoroughly lysed using Trizol for RNA extraction. RNA fragments within the size range of 26 34nt was separated using polyacrylamide gel electrophoresis without xxx and the corresponding bands were collected. The recovered bands were subjected to ligation reaction where a preadenylylated and three prime blocked linker five prime rApp CTGTAGGCACCATCAAT NH2 three prime was attached to the three prime end of the RNA. The ligated products were then recovered by performing polyacrylamide gel electrophoresis. Subsequently the recovered RNA fragments were reverse transcribed using a reverse primer to obtain extended cDNA molecules. The purified cDNA was subjected to circularization by using CircLigase Epicentre CL4111K resulting in the formation of circular cDNA molecules. To remove residual rRNA components in the cDNA a method involving hybridization with complementary primers specific to rRNA and subsequent heat denaturation was employed. The remaining circularized cDNA was then amplified through PCR and barcode sequences were incorporated.   OTHER TRANSCRIPTOMIC other PAIRED ILLUMINA Illumina NovaSeq 6000   SRP503769     un_2.raw.1.fastq.gz un_2.raw.2.fastq.gz fastq fastq 12132018896.0 40172248.0 GSM8228798 r1 0:151 1:151 A:2225485331;C:2027579165;G:5953072417;T:1925141784;N:740199 151 151     2225485331 2027579165 5953072417 1925141784 740199 SRX24354548 SRS21112316 SRA1852128 ShanDong University ShanDong University                       T T mates < 9% mapping rate illumina novaseq_era 3prime random_priming unknown bulk unknown unknown   China 2024-04-24 Gastrula Embryo Whole Organism All anatomical structures
31950 31950 SRR28790257 SRX24354547 SRS21112315 SRP503769 PRJNA1104180 Double stranded RNA triggers a distinct integrated stress response in the early embryo [RNA seq Ribo seq] GSE265771 Other Double stranded RNA dsRNA is associated with virus infections and is present as by products during the transcription of synthetic mRNA which has been widely used in gene gain of function studies and serves as a core component in emerging mRNA based therapies1 4. The presence of dsRNA in host cells induces an integrated stress response that functions to prevent virus replication and infection5 6. Unlike differentiated cells undifferentiated cells adopt a distinct defense strategy against RNA virus infection7 but the mechanism is unclear. We show a previously unidentified response triggered by dsRNA in the early embryo. Although dsRNA causes a global protein translation inhibition in a PKR eIF2a independent manner and leads to developmental delay and cell necrosis it also strongly induces p53 activation which then upregulates Interferon Stimulated Genes independently of interferon ligands. Importantly we demonstrate that the burst of p53 signaling dose not result in cell death but functions as a protective mechanism against deleterious translation blockage by slowing down global protein degradation via ISGylation. Our work has identified a distinct dsRNA induced stress response in the embryo reflecting an ancient innate immune memory before the establishment of the IFN system. It also raises the provocative question as to the original protective role of p53 during evolution. Overall design: To characterize the distribution of ribosomes on mRNA we performed Ribosome profiling Ribo seq analysis with RNA seq on dsRNA injected embryos.       Zebrafish Riboseq low dose uninj1 GSM8228797   source name:whole embryo|tissue:whole embryo|developmental stage:50% epiboly stage|cell type:embryonic cell|genotype:wild type|treatment:uninjected|geo loc name:missing|collection date:missing Zebrafish Riboseq low dose uninj1 The library underwent quality control assessment and was subjected to Illumina Novaseq 6000 sequencing. Bio informatics analysis of RIBOseq profilling was performed to analyze the library data. Assembly: GRCz10 Supplementary files format and content: Excel file includes raw counts for each Sample Library strategy: Ribo seq whole embryo   Both control group and dsRNA injected embryos at xxx hpf were thoroughly lysed on ice using lysis buffer.The lysate was then centrifuged at 12000g at 4°C for 10 minutes to remove cellular components including undigested material such as cell nuclei.Then RNaseI was added to the supernatant and incubated at 25°C for 30 minutes to digest RNA while the RNA fragments protected by ribosomes were preserved. Subsequently an RNA inhibitor was added to terminate the digestion reaction. The lysate was subjected to sucrose density gradient centrifugation and fractions containing single peaks at 260nm wavelength around the 80S monosome region were collected. The collected mixture was then thoroughly lysed using Trizol for RNA extraction. RNA fragments within the size range of 26 34nt was separated using polyacrylamide gel electrophoresis without xxx and the corresponding bands were collected. The recovered bands were subjected to ligation reaction where a preadenylylated and 3’ blocked linker 5’ rApp CTGTAGGCACCATCAAT NH2 3’ was attached to the three prime end of the RNA. The ligated products were then recovered by performing polyacrylamide gel electrophoresis. Subsequently the recovered RNA fragments were reverse transcribed using a reverse primer to obtain extended cDNA molecules. The purified cDNA was subjected to circularization by using CircLigase Epicentre CL4111K resulting in the formation of circular cDNA molecules. To remove residual rRNA components in the cDNA a method involving hybridization with complementary primers specific to rRNA and subsequent heat denaturation was employed. The remaining circularized cDNA was then amplified through PCR and barcode sequences were incorporated.   tissue:whole embryo|developmental stage:50% epiboly stage|cell type:embryonic cell|genotype:wild type|treatment:uninjected GSM8228797 GSM8228797: Zebrafish Riboseq low dose uninj1; Danio rerio; OTHER GSM8228797 r1 GSM8228797 1 Both control group and dsRNA injected embryos at xxx hpf were thoroughly lysed on ice using lysis buffer.The lysate was then centrifuged at 12000g at 4°C for 10 minutes to remove cellular components including undigested material such as cell nuclei.Then RNaseI was added to the supernatant and incubated at 25°C for 30 minutes to digest RNA while the RNA fragments protected by ribosomes were preserved. Subsequently an RNA inhibitor was added to terminate the digestion reaction. The lysate was subjected to sucrose density gradient centrifugation and fractions containing single peaks at 260nm wavelength around the 80S monosome region were collected. The collected mixture was then thoroughly lysed using Trizol for RNA extraction. RNA fragments within the size range of 26 34nt was separated using polyacrylamide gel electrophoresis without xxx and the corresponding bands were collected. The recovered bands were subjected to ligation reaction where a preadenylylated and three prime blocked linker five prime rApp CTGTAGGCACCATCAAT NH2 three prime was attached to the three prime end of the RNA. The ligated products were then recovered by performing polyacrylamide gel electrophoresis. Subsequently the recovered RNA fragments were reverse transcribed using a reverse primer to obtain extended cDNA molecules. The purified cDNA was subjected to circularization by using CircLigase Epicentre CL4111K resulting in the formation of circular cDNA molecules. To remove residual rRNA components in the cDNA a method involving hybridization with complementary primers specific to rRNA and subsequent heat denaturation was employed. The remaining circularized cDNA was then amplified through PCR and barcode sequences were incorporated.   OTHER TRANSCRIPTOMIC other PAIRED ILLUMINA Illumina NovaSeq 6000   SRP503769     un_1.raw.1.fastq.gz un_1.raw.2.fastq.gz fastq fastq 10423454802.0 34514751.0 GSM8228797 r1 0:151 1:151 A:1931094519;C:1788894999;G:5028533789;T:1674294221;N:637274 151 151     1931094519 1788894999 5028533789 1674294221 637274 SRX24354547 SRS21112315 SRA1852128 ShanDong University ShanDong University                       T T mates < 9% mapping rate illumina novaseq_era 3prime random_priming unknown bulk unknown unknown   China 2024-04-24 Gastrula Embryo Whole Organism All anatomical structures
32725 32725 SRR29398864 SRX24912671 SRS21618605 SRP513754 PRJNA1123686 Cell state transitions are decoupled from cell division during early embryo development [I] GSE269784 Other As tissues develop cells divide and differentiate concurrently. Conflicting evidence shows that cell division is either dispensable or required for formation of cell types. To determine the role of cell division in differentiation we arrested the cell cycle in zebrafish embryos using two independent approaches and profiled them at single cell resolution. We show that cell division is dispensable for differentiation of all embryonic tissues during initial cell type differentiation from early gastrulation to the end of segmentation. However in the absence of cell division differentiation slows down in some cell types and cells exhibit global stress responses. While differentiation is robust to blocking cell division the proportions of cells across cell states are not but show evidence of partial compensation. This work clarifies our understanding of the role of cell division in development and showcases the utility of combining embryo wide perturbations with single cell RNA sequencing to uncover the role of common biological processes across multiple tissues. Overall design: We arrested the cell cycle in zebrafish embryos at 6 hpf using two independent approaches: a chemical approach hydroxyurea and aphidicolin HUA and a genetic approach involving a loss of function mutation in the gene emi1 allele hi2648. We tracked differentiation dynamics using single cell RNA sequencing scRNA seq on embryos spanning 6–24 hpf for HUA treated and control embryos and at 24 hpf for emi1 mutant embryos. Overall we have collected multiple replicates for control embryos ABs at 6 8 10 14 18 21 hpf and 24 hpf for HUA treated at 8 10 14 hpf and 24 hpf and for emi1 mutants at 24 hpf. Details about the samples can be found in the supplementary file "sample information.txt"   pubmed:37546736   Perturbation experiment 3 24 hpf biological replicate 1 to 3 technical replicate 2 3 GSM8327220   source name:whole embryo|tissue:whole embryo|treatment:control and emi1 homozygous mutants|geo loc name:missing|collection date:missing Perturbation experiment 3 24 hpf biological replicate 1 to 3 technical replicate 2 3 Multiple samples are pooled for each sequencing run. Raw FASTQ were prepared from Illumina bcl files in a format compatible with the inDrops.py pipeline. Each nextseq run results in 16 FASTQ files 4 lanes x 4 reads per lane. For some pooled libraries sequencing was run twice to get more UMIs per cell and hence we have deposited 16x2 = 32 raw files for those sequencing samples. The illumina library indices of different samples in a run are provided in the meta data. These can be used to demultiplex the raw file into separate libraries. The read files from an inDrops run have the following content: * R1 001.fastq.gz : contains the three prime UTR cDNA read * R2 001.fastq.gz : contains the first half of the cell barcode * R3 001.fastq.gz : contains the library index for demultiplexing libraries * R4 001.fastq.gz : contains the second half of the barcode and the UMI. All subsequent processing steps from FASTQ files to count matrixes were performed using the custom pipeline for inDrops data analysis github.com/indrops. Single cell transcriptomes were barcoded using inDrops Klein et al Cell 2015. Standard transcriptome RNA seq libraries were processed as reported in Zilionis et al. Nature Protocol 2016 using inDrops v3 protocol. The transcriptome libraries were sequenced on reads Illumina NextSeq 500. Libraries used standard Illumina sequencing primers and 61 cycles for Read1 14 cycles for Read2 8 cycles each for IndexRead1 and IndexRead2. Raw fastq files was processed using inDrops.py pipeline github.com/indrops/indrops. Sequenced reads were mapped to a zebrafish reference transcriptome built from the zebrafish GRCz10 genome assembly Assembly Accession: GCF 000002035.5 using bowtie version 1.1.143. To obtain the final counts matrix used for data analysis total count filters were applied as described in Kukreja et. al. 2024 method section "Single cell RNA seq Data preprocessing" Assembly: GRCz10 genome assembly Assembly Accession: GCF 000002035.5 Supplementary files format and content: Raw counts tsv.gz: cell ba… whole embryo Zebrafish embryos were arrested for cell cycle using two complementary approaches. S phase cell cycle arrest was induced using a cocktail of drugs – hydroxyurea Sigma Aldrich H8627 5G at 20 mM and aphidicolin Sigma Aldrich A0781 5MG at 150 µM concentration in 1% dimethyl sulfoxide DMSO in egg water. G2/M phase arrest was induced by crossing heterozygous emi1 wt/mut zebrafish to generate homozygous emi1 mut/mut embryos referred as hi2648 in the manuscript. Homozygous mutants with cell cycle arrest were screened at 24 hpf as they have very clear phenotype by this time – these embryos are smaller and have bent tails and the heterozygous mutants and wildtype are indistinguishable. Zebrafish embryos were dechorionated using 1mg/mL Pronase Sigma P5147 1G for 5 7 minutes followed by washing in egg water. Embryos were dissociated as previously described in Wagner et. al. Science 2018. Briefly embryos were dissociated in 0.5mL LoBind microcentrifuge tubes that had been precoated with 10% w/v BSA for about 15 minutes at room temperature. They were homogenized in 1XDPBS/1% w/v BSA for 6 hpf to 10 hpf embryos early time points and in 500 µL FACSmax cell dissociation solution Genlantis T200100 for 14 hpf to 24 hpf embryos late time points. Cells were then filtered through a 40µm cell strainer mesh Fisher 352340 and centrifuged in a swinging bucket rotor at 200g for 1 minute early time points or 300g for 5 minutes late time points. Cells were then washed in 1XDPBS/1%BSA using the same centrifuge settings. They were then resuspended in 1XDPBS/0.5%BSA/18% optiprep density medium Sigma D1556 250ML. Cell concentration was manually quantified using hemocytometer and adjusted to a final concentration of 100 000 cells/mL before encapsulation. Library construction as detailed in Zilionis R. et al. 2016 Nature Protocols. Single cell barcoding and sequencing was done using droplet microfluidics also described in the same paper. AB wild type strains were used for all HUA perturbation experiments. Zebrafish mutant line hi2648 a mutant for the gene emi1 was kindly gifted by Dr. Jennifer Rhodes. Embryos were developed within the ambient temperature of Zebrafish development. Time of fertilization at 28.5 C was used to stage each clutch and this was confirmed using morphological features of the embryos. All zebrafish were housed in a facility overseen by the Harvard Medical Area Standing Committee on Animals our IACUC which performs regular inspections and under which we have an approved protocol for all animal procedures. tissue:whole embryo|treatment:control and emi1 homozygous mutants GSM8327220 GSM8327220: Perturbation experiment 3 24 hpf biological replicate 1 to 3 technical replicate 2 3; Danio rerio; OTHER GSM8327220 r1 GSM8327220 1 Zebrafish embryos were dechorionated using 1mg/mL Pronase Sigma P5147 1G for 5 7 minutes followed by washing in egg water. Embryos were dissociated as previously described in Wagner et. al. Science 2018. Briefly embryos were dissociated in 0.5mL LoBind microcentrifuge tubes that had been precoated with 10% w/v BSA for about 15 minutes at room temperature. They were homogenized in 1XDPBS/1% w/v BSA for 6 hpf to 10 hpf embryos early time points and in 500 µL FACSmax cell dissociation solution Genlantis T200100 for 14 hpf to 24 hpf embryos late time points. Cells were then filtered through a 40µm cell strainer mesh Fisher 352340 and centrifuged in a swinging bucket rotor at 200g for 1 minute early time points or 300g for 5 minutes late time points. Cells were then washed in 1XDPBS/1%BSA using the same centrifuge settings. They were then resuspended in 1XDPBS/0.5%BSA/18% optiprep density medium Sigma D1556 250ML. Cell concentration was manually quantified using hemocytometer and adjusted to a final concentration of 100 000 cells/mL before encapsulation. Library construction as detailed in Zilionis R. et al. 2016 Nature Protocols. Single cell barcoding and sequencing was done using droplet microfluidics also described in the same paper.   OTHER TRANSCRIPTOMIC other PAIRED ILLUMINA NextSeq 500   SRP513754     pert_exp3_brep1_2_3_trep2_3_S0_L001_R1_001.fastq.gz pert_exp3_brep1_2_3_trep2_3_S0_L001_R2_001.fastq.gz pert_exp3_brep1_2_3_trep2_3_S0_L001_R3_001.fastq.gz pert_exp3_brep1_2_3_trep2_3_S0_L001_R4_001.fastq.gz fastq fastq fastq fastq 10196913181.0 112053991.0 GSM8327220 r1 0:61 1:8 2:8 3:14 A:1907833453;C:1411947502;G:1388620349;T:2126120065;N:772082 61 8 8 14 1907833453 1411947502 1388620349 2126120065 772082 SRX24912671 SRS21618605 SRA1898111 Harvard University Harvard University                       B   usable mapping rate illumina nextseq unknown other trueseq sc single_cell_droplet indrops   United States 2024-06-13 Multi-stage Embryo Whole Organism All anatomical structures
32726 32726 SRR29398865 SRX24912671 SRS21618605 SRP513754 PRJNA1123686 Cell state transitions are decoupled from cell division during early embryo development [I] GSE269784 Other As tissues develop cells divide and differentiate concurrently. Conflicting evidence shows that cell division is either dispensable or required for formation of cell types. To determine the role of cell division in differentiation we arrested the cell cycle in zebrafish embryos using two independent approaches and profiled them at single cell resolution. We show that cell division is dispensable for differentiation of all embryonic tissues during initial cell type differentiation from early gastrulation to the end of segmentation. However in the absence of cell division differentiation slows down in some cell types and cells exhibit global stress responses. While differentiation is robust to blocking cell division the proportions of cells across cell states are not but show evidence of partial compensation. This work clarifies our understanding of the role of cell division in development and showcases the utility of combining embryo wide perturbations with single cell RNA sequencing to uncover the role of common biological processes across multiple tissues. Overall design: We arrested the cell cycle in zebrafish embryos at 6 hpf using two independent approaches: a chemical approach hydroxyurea and aphidicolin HUA and a genetic approach involving a loss of function mutation in the gene emi1 allele hi2648. We tracked differentiation dynamics using single cell RNA sequencing scRNA seq on embryos spanning 6–24 hpf for HUA treated and control embryos and at 24 hpf for emi1 mutant embryos. Overall we have collected multiple replicates for control embryos ABs at 6 8 10 14 18 21 hpf and 24 hpf for HUA treated at 8 10 14 hpf and 24 hpf and for emi1 mutants at 24 hpf. Details about the samples can be found in the supplementary file "sample information.txt"   pubmed:37546736   Perturbation experiment 3 24 hpf biological replicate 1 to 3 technical replicate 2 3 GSM8327220   source name:whole embryo|tissue:whole embryo|treatment:control and emi1 homozygous mutants|geo loc name:missing|collection date:missing Perturbation experiment 3 24 hpf biological replicate 1 to 3 technical replicate 2 3 Multiple samples are pooled for each sequencing run. Raw FASTQ were prepared from Illumina bcl files in a format compatible with the inDrops.py pipeline. Each nextseq run results in 16 FASTQ files 4 lanes x 4 reads per lane. For some pooled libraries sequencing was run twice to get more UMIs per cell and hence we have deposited 16x2 = 32 raw files for those sequencing samples. The illumina library indices of different samples in a run are provided in the meta data. These can be used to demultiplex the raw file into separate libraries. The read files from an inDrops run have the following content: * R1 001.fastq.gz : contains the three prime UTR cDNA read * R2 001.fastq.gz : contains the first half of the cell barcode * R3 001.fastq.gz : contains the library index for demultiplexing libraries * R4 001.fastq.gz : contains the second half of the barcode and the UMI. All subsequent processing steps from FASTQ files to count matrixes were performed using the custom pipeline for inDrops data analysis github.com/indrops. Single cell transcriptomes were barcoded using inDrops Klein et al Cell 2015. Standard transcriptome RNA seq libraries were processed as reported in Zilionis et al. Nature Protocol 2016 using inDrops v3 protocol. The transcriptome libraries were sequenced on reads Illumina NextSeq 500. Libraries used standard Illumina sequencing primers and 61 cycles for Read1 14 cycles for Read2 8 cycles each for IndexRead1 and IndexRead2. Raw fastq files was processed using inDrops.py pipeline github.com/indrops/indrops. Sequenced reads were mapped to a zebrafish reference transcriptome built from the zebrafish GRCz10 genome assembly Assembly Accession: GCF 000002035.5 using bowtie version 1.1.143. To obtain the final counts matrix used for data analysis total count filters were applied as described in Kukreja et. al. 2024 method section "Single cell RNA seq Data preprocessing" Assembly: GRCz10 genome assembly Assembly Accession: GCF 000002035.5 Supplementary files format and content: Raw counts tsv.gz: cell ba… whole embryo Zebrafish embryos were arrested for cell cycle using two complementary approaches. S phase cell cycle arrest was induced using a cocktail of drugs – hydroxyurea Sigma Aldrich H8627 5G at 20 mM and aphidicolin Sigma Aldrich A0781 5MG at 150 µM concentration in 1% dimethyl sulfoxide DMSO in egg water. G2/M phase arrest was induced by crossing heterozygous emi1 wt/mut zebrafish to generate homozygous emi1 mut/mut embryos referred as hi2648 in the manuscript. Homozygous mutants with cell cycle arrest were screened at 24 hpf as they have very clear phenotype by this time – these embryos are smaller and have bent tails and the heterozygous mutants and wildtype are indistinguishable. Zebrafish embryos were dechorionated using 1mg/mL Pronase Sigma P5147 1G for 5 7 minutes followed by washing in egg water. Embryos were dissociated as previously described in Wagner et. al. Science 2018. Briefly embryos were dissociated in 0.5mL LoBind microcentrifuge tubes that had been precoated with 10% w/v BSA for about 15 minutes at room temperature. They were homogenized in 1XDPBS/1% w/v BSA for 6 hpf to 10 hpf embryos early time points and in 500 µL FACSmax cell dissociation solution Genlantis T200100 for 14 hpf to 24 hpf embryos late time points. Cells were then filtered through a 40µm cell strainer mesh Fisher 352340 and centrifuged in a swinging bucket rotor at 200g for 1 minute early time points or 300g for 5 minutes late time points. Cells were then washed in 1XDPBS/1%BSA using the same centrifuge settings. They were then resuspended in 1XDPBS/0.5%BSA/18% optiprep density medium Sigma D1556 250ML. Cell concentration was manually quantified using hemocytometer and adjusted to a final concentration of 100 000 cells/mL before encapsulation. Library construction as detailed in Zilionis R. et al. 2016 Nature Protocols. Single cell barcoding and sequencing was done using droplet microfluidics also described in the same paper. AB wild type strains were used for all HUA perturbation experiments. Zebrafish mutant line hi2648 a mutant for the gene emi1 was kindly gifted by Dr. Jennifer Rhodes. Embryos were developed within the ambient temperature of Zebrafish development. Time of fertilization at 28.5 C was used to stage each clutch and this was confirmed using morphological features of the embryos. All zebrafish were housed in a facility overseen by the Harvard Medical Area Standing Committee on Animals our IACUC which performs regular inspections and under which we have an approved protocol for all animal procedures. tissue:whole embryo|treatment:control and emi1 homozygous mutants GSM8327220 GSM8327220: Perturbation experiment 3 24 hpf biological replicate 1 to 3 technical replicate 2 3; Danio rerio; OTHER GSM8327220 r1 GSM8327220 1 Zebrafish embryos were dechorionated using 1mg/mL Pronase Sigma P5147 1G for 5 7 minutes followed by washing in egg water. Embryos were dissociated as previously described in Wagner et. al. Science 2018. Briefly embryos were dissociated in 0.5mL LoBind microcentrifuge tubes that had been precoated with 10% w/v BSA for about 15 minutes at room temperature. They were homogenized in 1XDPBS/1% w/v BSA for 6 hpf to 10 hpf embryos early time points and in 500 µL FACSmax cell dissociation solution Genlantis T200100 for 14 hpf to 24 hpf embryos late time points. Cells were then filtered through a 40µm cell strainer mesh Fisher 352340 and centrifuged in a swinging bucket rotor at 200g for 1 minute early time points or 300g for 5 minutes late time points. Cells were then washed in 1XDPBS/1%BSA using the same centrifuge settings. They were then resuspended in 1XDPBS/0.5%BSA/18% optiprep density medium Sigma D1556 250ML. Cell concentration was manually quantified using hemocytometer and adjusted to a final concentration of 100 000 cells/mL before encapsulation. Library construction as detailed in Zilionis R. et al. 2016 Nature Protocols. Single cell barcoding and sequencing was done using droplet microfluidics also described in the same paper.   OTHER TRANSCRIPTOMIC other PAIRED ILLUMINA NextSeq 500   SRP513754     pert_exp3_brep1_2_3_trep2_3_S0_L002_R1_001.fastq.gz pert_exp3_brep1_2_3_trep2_3_S0_L002_R2_001.fastq.gz pert_exp3_brep1_2_3_trep2_3_S0_L002_R3_001.fastq.gz pert_exp3_brep1_2_3_trep2_3_S0_L002_R4_001.fastq.gz fastq fastq fastq fastq 10073565229.0 110698519.0 GSM8327220 r2 0:61 1:8 2:8 3:14 A:1882668771;C:1393477449;G:1377270278;T:2098291783;N:901378 61 8 8 14 1882668771 1393477449 1377270278 2098291783 901378 SRX24912671 SRS21618605 SRA1898111 Harvard University Harvard University                       B   usable mapping rate illumina nextseq unknown other trueseq sc single_cell_droplet indrops   United States 2024-06-13 Multi-stage Embryo Whole Organism All anatomical structures
32727 32727 SRR29398866 SRX24912671 SRS21618605 SRP513754 PRJNA1123686 Cell state transitions are decoupled from cell division during early embryo development [I] GSE269784 Other As tissues develop cells divide and differentiate concurrently. Conflicting evidence shows that cell division is either dispensable or required for formation of cell types. To determine the role of cell division in differentiation we arrested the cell cycle in zebrafish embryos using two independent approaches and profiled them at single cell resolution. We show that cell division is dispensable for differentiation of all embryonic tissues during initial cell type differentiation from early gastrulation to the end of segmentation. However in the absence of cell division differentiation slows down in some cell types and cells exhibit global stress responses. While differentiation is robust to blocking cell division the proportions of cells across cell states are not but show evidence of partial compensation. This work clarifies our understanding of the role of cell division in development and showcases the utility of combining embryo wide perturbations with single cell RNA sequencing to uncover the role of common biological processes across multiple tissues. Overall design: We arrested the cell cycle in zebrafish embryos at 6 hpf using two independent approaches: a chemical approach hydroxyurea and aphidicolin HUA and a genetic approach involving a loss of function mutation in the gene emi1 allele hi2648. We tracked differentiation dynamics using single cell RNA sequencing scRNA seq on embryos spanning 6–24 hpf for HUA treated and control embryos and at 24 hpf for emi1 mutant embryos. Overall we have collected multiple replicates for control embryos ABs at 6 8 10 14 18 21 hpf and 24 hpf for HUA treated at 8 10 14 hpf and 24 hpf and for emi1 mutants at 24 hpf. Details about the samples can be found in the supplementary file "sample information.txt"   pubmed:37546736   Perturbation experiment 3 24 hpf biological replicate 1 to 3 technical replicate 2 3 GSM8327220   source name:whole embryo|tissue:whole embryo|treatment:control and emi1 homozygous mutants|geo loc name:missing|collection date:missing Perturbation experiment 3 24 hpf biological replicate 1 to 3 technical replicate 2 3 Multiple samples are pooled for each sequencing run. Raw FASTQ were prepared from Illumina bcl files in a format compatible with the inDrops.py pipeline. Each nextseq run results in 16 FASTQ files 4 lanes x 4 reads per lane. For some pooled libraries sequencing was run twice to get more UMIs per cell and hence we have deposited 16x2 = 32 raw files for those sequencing samples. The illumina library indices of different samples in a run are provided in the meta data. These can be used to demultiplex the raw file into separate libraries. The read files from an inDrops run have the following content: * R1 001.fastq.gz : contains the three prime UTR cDNA read * R2 001.fastq.gz : contains the first half of the cell barcode * R3 001.fastq.gz : contains the library index for demultiplexing libraries * R4 001.fastq.gz : contains the second half of the barcode and the UMI. All subsequent processing steps from FASTQ files to count matrixes were performed using the custom pipeline for inDrops data analysis github.com/indrops. Single cell transcriptomes were barcoded using inDrops Klein et al Cell 2015. Standard transcriptome RNA seq libraries were processed as reported in Zilionis et al. Nature Protocol 2016 using inDrops v3 protocol. The transcriptome libraries were sequenced on reads Illumina NextSeq 500. Libraries used standard Illumina sequencing primers and 61 cycles for Read1 14 cycles for Read2 8 cycles each for IndexRead1 and IndexRead2. Raw fastq files was processed using inDrops.py pipeline github.com/indrops/indrops. Sequenced reads were mapped to a zebrafish reference transcriptome built from the zebrafish GRCz10 genome assembly Assembly Accession: GCF 000002035.5 using bowtie version 1.1.143. To obtain the final counts matrix used for data analysis total count filters were applied as described in Kukreja et. al. 2024 method section "Single cell RNA seq Data preprocessing" Assembly: GRCz10 genome assembly Assembly Accession: GCF 000002035.5 Supplementary files format and content: Raw counts tsv.gz: cell ba… whole embryo Zebrafish embryos were arrested for cell cycle using two complementary approaches. S phase cell cycle arrest was induced using a cocktail of drugs – hydroxyurea Sigma Aldrich H8627 5G at 20 mM and aphidicolin Sigma Aldrich A0781 5MG at 150 µM concentration in 1% dimethyl sulfoxide DMSO in egg water. G2/M phase arrest was induced by crossing heterozygous emi1 wt/mut zebrafish to generate homozygous emi1 mut/mut embryos referred as hi2648 in the manuscript. Homozygous mutants with cell cycle arrest were screened at 24 hpf as they have very clear phenotype by this time – these embryos are smaller and have bent tails and the heterozygous mutants and wildtype are indistinguishable. Zebrafish embryos were dechorionated using 1mg/mL Pronase Sigma P5147 1G for 5 7 minutes followed by washing in egg water. Embryos were dissociated as previously described in Wagner et. al. Science 2018. Briefly embryos were dissociated in 0.5mL LoBind microcentrifuge tubes that had been precoated with 10% w/v BSA for about 15 minutes at room temperature. They were homogenized in 1XDPBS/1% w/v BSA for 6 hpf to 10 hpf embryos early time points and in 500 µL FACSmax cell dissociation solution Genlantis T200100 for 14 hpf to 24 hpf embryos late time points. Cells were then filtered through a 40µm cell strainer mesh Fisher 352340 and centrifuged in a swinging bucket rotor at 200g for 1 minute early time points or 300g for 5 minutes late time points. Cells were then washed in 1XDPBS/1%BSA using the same centrifuge settings. They were then resuspended in 1XDPBS/0.5%BSA/18% optiprep density medium Sigma D1556 250ML. Cell concentration was manually quantified using hemocytometer and adjusted to a final concentration of 100 000 cells/mL before encapsulation. Library construction as detailed in Zilionis R. et al. 2016 Nature Protocols. Single cell barcoding and sequencing was done using droplet microfluidics also described in the same paper. AB wild type strains were used for all HUA perturbation experiments. Zebrafish mutant line hi2648 a mutant for the gene emi1 was kindly gifted by Dr. Jennifer Rhodes. Embryos were developed within the ambient temperature of Zebrafish development. Time of fertilization at 28.5 C was used to stage each clutch and this was confirmed using morphological features of the embryos. All zebrafish were housed in a facility overseen by the Harvard Medical Area Standing Committee on Animals our IACUC which performs regular inspections and under which we have an approved protocol for all animal procedures. tissue:whole embryo|treatment:control and emi1 homozygous mutants GSM8327220 GSM8327220: Perturbation experiment 3 24 hpf biological replicate 1 to 3 technical replicate 2 3; Danio rerio; OTHER GSM8327220 r1 GSM8327220 1 Zebrafish embryos were dechorionated using 1mg/mL Pronase Sigma P5147 1G for 5 7 minutes followed by washing in egg water. Embryos were dissociated as previously described in Wagner et. al. Science 2018. Briefly embryos were dissociated in 0.5mL LoBind microcentrifuge tubes that had been precoated with 10% w/v BSA for about 15 minutes at room temperature. They were homogenized in 1XDPBS/1% w/v BSA for 6 hpf to 10 hpf embryos early time points and in 500 µL FACSmax cell dissociation solution Genlantis T200100 for 14 hpf to 24 hpf embryos late time points. Cells were then filtered through a 40µm cell strainer mesh Fisher 352340 and centrifuged in a swinging bucket rotor at 200g for 1 minute early time points or 300g for 5 minutes late time points. Cells were then washed in 1XDPBS/1%BSA using the same centrifuge settings. They were then resuspended in 1XDPBS/0.5%BSA/18% optiprep density medium Sigma D1556 250ML. Cell concentration was manually quantified using hemocytometer and adjusted to a final concentration of 100 000 cells/mL before encapsulation. Library construction as detailed in Zilionis R. et al. 2016 Nature Protocols. Single cell barcoding and sequencing was done using droplet microfluidics also described in the same paper.   OTHER TRANSCRIPTOMIC other PAIRED ILLUMINA NextSeq 500   SRP513754     pert_exp3_brep1_2_3_trep2_3_S0_L003_R1_001.fastq.gz pert_exp3_brep1_2_3_trep2_3_S0_L003_R2_001.fastq.gz pert_exp3_brep1_2_3_trep2_3_S0_L003_R3_001.fastq.gz pert_exp3_brep1_2_3_trep2_3_S0_L003_R4_001.fastq.gz fastq fastq fastq fastq 10334862265.0 113569915.0 GSM8327220 r3 0:61 1:8 2:8 3:14 A:1932620835;C:1429967805;G:1408958497;T:2155416872;N:800806 61 8 8 14 1932620835 1429967805 1408958497 2155416872 800806 SRX24912671 SRS21618605 SRA1898111 Harvard University Harvard University                       B   usable mapping rate illumina nextseq unknown other trueseq sc single_cell_droplet indrops   United States 2024-06-13 Multi-stage Embryo Whole Organism All anatomical structures
32728 32728 SRR29398867 SRX24912671 SRS21618605 SRP513754 PRJNA1123686 Cell state transitions are decoupled from cell division during early embryo development [I] GSE269784 Other As tissues develop cells divide and differentiate concurrently. Conflicting evidence shows that cell division is either dispensable or required for formation of cell types. To determine the role of cell division in differentiation we arrested the cell cycle in zebrafish embryos using two independent approaches and profiled them at single cell resolution. We show that cell division is dispensable for differentiation of all embryonic tissues during initial cell type differentiation from early gastrulation to the end of segmentation. However in the absence of cell division differentiation slows down in some cell types and cells exhibit global stress responses. While differentiation is robust to blocking cell division the proportions of cells across cell states are not but show evidence of partial compensation. This work clarifies our understanding of the role of cell division in development and showcases the utility of combining embryo wide perturbations with single cell RNA sequencing to uncover the role of common biological processes across multiple tissues. Overall design: We arrested the cell cycle in zebrafish embryos at 6 hpf using two independent approaches: a chemical approach hydroxyurea and aphidicolin HUA and a genetic approach involving a loss of function mutation in the gene emi1 allele hi2648. We tracked differentiation dynamics using single cell RNA sequencing scRNA seq on embryos spanning 6–24 hpf for HUA treated and control embryos and at 24 hpf for emi1 mutant embryos. Overall we have collected multiple replicates for control embryos ABs at 6 8 10 14 18 21 hpf and 24 hpf for HUA treated at 8 10 14 hpf and 24 hpf and for emi1 mutants at 24 hpf. Details about the samples can be found in the supplementary file "sample information.txt"   pubmed:37546736   Perturbation experiment 3 24 hpf biological replicate 1 to 3 technical replicate 2 3 GSM8327220   source name:whole embryo|tissue:whole embryo|treatment:control and emi1 homozygous mutants|geo loc name:missing|collection date:missing Perturbation experiment 3 24 hpf biological replicate 1 to 3 technical replicate 2 3 Multiple samples are pooled for each sequencing run. Raw FASTQ were prepared from Illumina bcl files in a format compatible with the inDrops.py pipeline. Each nextseq run results in 16 FASTQ files 4 lanes x 4 reads per lane. For some pooled libraries sequencing was run twice to get more UMIs per cell and hence we have deposited 16x2 = 32 raw files for those sequencing samples. The illumina library indices of different samples in a run are provided in the meta data. These can be used to demultiplex the raw file into separate libraries. The read files from an inDrops run have the following content: * R1 001.fastq.gz : contains the three prime UTR cDNA read * R2 001.fastq.gz : contains the first half of the cell barcode * R3 001.fastq.gz : contains the library index for demultiplexing libraries * R4 001.fastq.gz : contains the second half of the barcode and the UMI. All subsequent processing steps from FASTQ files to count matrixes were performed using the custom pipeline for inDrops data analysis github.com/indrops. Single cell transcriptomes were barcoded using inDrops Klein et al Cell 2015. Standard transcriptome RNA seq libraries were processed as reported in Zilionis et al. Nature Protocol 2016 using inDrops v3 protocol. The transcriptome libraries were sequenced on reads Illumina NextSeq 500. Libraries used standard Illumina sequencing primers and 61 cycles for Read1 14 cycles for Read2 8 cycles each for IndexRead1 and IndexRead2. Raw fastq files was processed using inDrops.py pipeline github.com/indrops/indrops. Sequenced reads were mapped to a zebrafish reference transcriptome built from the zebrafish GRCz10 genome assembly Assembly Accession: GCF 000002035.5 using bowtie version 1.1.143. To obtain the final counts matrix used for data analysis total count filters were applied as described in Kukreja et. al. 2024 method section "Single cell RNA seq Data preprocessing" Assembly: GRCz10 genome assembly Assembly Accession: GCF 000002035.5 Supplementary files format and content: Raw counts tsv.gz: cell ba… whole embryo Zebrafish embryos were arrested for cell cycle using two complementary approaches. S phase cell cycle arrest was induced using a cocktail of drugs – hydroxyurea Sigma Aldrich H8627 5G at 20 mM and aphidicolin Sigma Aldrich A0781 5MG at 150 µM concentration in 1% dimethyl sulfoxide DMSO in egg water. G2/M phase arrest was induced by crossing heterozygous emi1 wt/mut zebrafish to generate homozygous emi1 mut/mut embryos referred as hi2648 in the manuscript. Homozygous mutants with cell cycle arrest were screened at 24 hpf as they have very clear phenotype by this time – these embryos are smaller and have bent tails and the heterozygous mutants and wildtype are indistinguishable. Zebrafish embryos were dechorionated using 1mg/mL Pronase Sigma P5147 1G for 5 7 minutes followed by washing in egg water. Embryos were dissociated as previously described in Wagner et. al. Science 2018. Briefly embryos were dissociated in 0.5mL LoBind microcentrifuge tubes that had been precoated with 10% w/v BSA for about 15 minutes at room temperature. They were homogenized in 1XDPBS/1% w/v BSA for 6 hpf to 10 hpf embryos early time points and in 500 µL FACSmax cell dissociation solution Genlantis T200100 for 14 hpf to 24 hpf embryos late time points. Cells were then filtered through a 40µm cell strainer mesh Fisher 352340 and centrifuged in a swinging bucket rotor at 200g for 1 minute early time points or 300g for 5 minutes late time points. Cells were then washed in 1XDPBS/1%BSA using the same centrifuge settings. They were then resuspended in 1XDPBS/0.5%BSA/18% optiprep density medium Sigma D1556 250ML. Cell concentration was manually quantified using hemocytometer and adjusted to a final concentration of 100 000 cells/mL before encapsulation. Library construction as detailed in Zilionis R. et al. 2016 Nature Protocols. Single cell barcoding and sequencing was done using droplet microfluidics also described in the same paper. AB wild type strains were used for all HUA perturbation experiments. Zebrafish mutant line hi2648 a mutant for the gene emi1 was kindly gifted by Dr. Jennifer Rhodes. Embryos were developed within the ambient temperature of Zebrafish development. Time of fertilization at 28.5 C was used to stage each clutch and this was confirmed using morphological features of the embryos. All zebrafish were housed in a facility overseen by the Harvard Medical Area Standing Committee on Animals our IACUC which performs regular inspections and under which we have an approved protocol for all animal procedures. tissue:whole embryo|treatment:control and emi1 homozygous mutants GSM8327220 GSM8327220: Perturbation experiment 3 24 hpf biological replicate 1 to 3 technical replicate 2 3; Danio rerio; OTHER GSM8327220 r1 GSM8327220 1 Zebrafish embryos were dechorionated using 1mg/mL Pronase Sigma P5147 1G for 5 7 minutes followed by washing in egg water. Embryos were dissociated as previously described in Wagner et. al. Science 2018. Briefly embryos were dissociated in 0.5mL LoBind microcentrifuge tubes that had been precoated with 10% w/v BSA for about 15 minutes at room temperature. They were homogenized in 1XDPBS/1% w/v BSA for 6 hpf to 10 hpf embryos early time points and in 500 µL FACSmax cell dissociation solution Genlantis T200100 for 14 hpf to 24 hpf embryos late time points. Cells were then filtered through a 40µm cell strainer mesh Fisher 352340 and centrifuged in a swinging bucket rotor at 200g for 1 minute early time points or 300g for 5 minutes late time points. Cells were then washed in 1XDPBS/1%BSA using the same centrifuge settings. They were then resuspended in 1XDPBS/0.5%BSA/18% optiprep density medium Sigma D1556 250ML. Cell concentration was manually quantified using hemocytometer and adjusted to a final concentration of 100 000 cells/mL before encapsulation. Library construction as detailed in Zilionis R. et al. 2016 Nature Protocols. Single cell barcoding and sequencing was done using droplet microfluidics also described in the same paper.   OTHER TRANSCRIPTOMIC other PAIRED ILLUMINA NextSeq 500   SRP513754     pert_exp3_brep1_2_3_trep2_3_S0_L004_R1_001.fastq.gz pert_exp3_brep1_2_3_trep2_3_S0_L004_R2_001.fastq.gz pert_exp3_brep1_2_3_trep2_3_S0_L004_R3_001.fastq.gz pert_exp3_brep1_2_3_trep2_3_S0_L004_R4_001.fastq.gz fastq fastq fastq fastq 10120552533.0 111214863.0 GSM8327220 r4 0:61 1:8 2:8 3:14 A:1891306008;C:1401185707;G:1383554545;T:2107193563;N:866820 61 8 8 14 1891306008 1401185707 1383554545 2107193563 866820 SRX24912671 SRS21618605 SRA1898111 Harvard University Harvard University                       B   usable mapping rate illumina nextseq unknown other trueseq sc single_cell_droplet indrops   United States 2024-06-13 Multi-stage Embryo Whole Organism All anatomical structures
32729 32729 SRR29398868 SRX24912670 SRS21618604 SRP513754 PRJNA1123686 Cell state transitions are decoupled from cell division during early embryo development [I] GSE269784 Other As tissues develop cells divide and differentiate concurrently. Conflicting evidence shows that cell division is either dispensable or required for formation of cell types. To determine the role of cell division in differentiation we arrested the cell cycle in zebrafish embryos using two independent approaches and profiled them at single cell resolution. We show that cell division is dispensable for differentiation of all embryonic tissues during initial cell type differentiation from early gastrulation to the end of segmentation. However in the absence of cell division differentiation slows down in some cell types and cells exhibit global stress responses. While differentiation is robust to blocking cell division the proportions of cells across cell states are not but show evidence of partial compensation. This work clarifies our understanding of the role of cell division in development and showcases the utility of combining embryo wide perturbations with single cell RNA sequencing to uncover the role of common biological processes across multiple tissues. Overall design: We arrested the cell cycle in zebrafish embryos at 6 hpf using two independent approaches: a chemical approach hydroxyurea and aphidicolin HUA and a genetic approach involving a loss of function mutation in the gene emi1 allele hi2648. We tracked differentiation dynamics using single cell RNA sequencing scRNA seq on embryos spanning 6–24 hpf for HUA treated and control embryos and at 24 hpf for emi1 mutant embryos. Overall we have collected multiple replicates for control embryos ABs at 6 8 10 14 18 21 hpf and 24 hpf for HUA treated at 8 10 14 hpf and 24 hpf and for emi1 mutants at 24 hpf. Details about the samples can be found in the supplementary file "sample information.txt"   pubmed:37546736   Perturbation experiment 3 24 hpf biological replicate 1 to 3 technical replicate 1 GSM8327219   source name:whole embryo|tissue:whole embryo|treatment:control and emi1 homozygous mutants|geo loc name:missing|collection date:missing Perturbation experiment 3 24 hpf biological replicate 1 to 3 technical replicate 1 Multiple samples are pooled for each sequencing run. Raw FASTQ were prepared from Illumina bcl files in a format compatible with the inDrops.py pipeline. Each nextseq run results in 16 FASTQ files 4 lanes x 4 reads per lane. For some pooled libraries sequencing was run twice to get more UMIs per cell and hence we have deposited 16x2 = 32 raw files for those sequencing samples. The illumina library indices of different samples in a run are provided in the meta data. These can be used to demultiplex the raw file into separate libraries. The read files from an inDrops run have the following content: * R1 001.fastq.gz : contains the three prime UTR cDNA read * R2 001.fastq.gz : contains the first half of the cell barcode * R3 001.fastq.gz : contains the library index for demultiplexing libraries * R4 001.fastq.gz : contains the second half of the barcode and the UMI. All subsequent processing steps from FASTQ files to count matrixes were performed using the custom pipeline for inDrops data analysis github.com/indrops. Single cell transcriptomes were barcoded using inDrops Klein et al Cell 2015. Standard transcriptome RNA seq libraries were processed as reported in Zilionis et al. Nature Protocol 2016 using inDrops v3 protocol. The transcriptome libraries were sequenced on reads Illumina NextSeq 500. Libraries used standard Illumina sequencing primers and 61 cycles for Read1 14 cycles for Read2 8 cycles each for IndexRead1 and IndexRead2. Raw fastq files was processed using inDrops.py pipeline github.com/indrops/indrops. Sequenced reads were mapped to a zebrafish reference transcriptome built from the zebrafish GRCz10 genome assembly Assembly Accession: GCF 000002035.5 using bowtie version 1.1.143. To obtain the final counts matrix used for data analysis total count filters were applied as described in Kukreja et. al. 2024 method section "Single cell RNA seq Data preprocessing" Assembly: GRCz10 genome assembly Assembly Accession: GCF 000002035.5 Supplementary files format and content: Raw counts tsv.gz: cell ba… whole embryo Zebrafish embryos were arrested for cell cycle using two complementary approaches. S phase cell cycle arrest was induced using a cocktail of drugs – hydroxyurea Sigma Aldrich H8627 5G at 20 mM and aphidicolin Sigma Aldrich A0781 5MG at 150 µM concentration in 1% dimethyl sulfoxide DMSO in egg water. G2/M phase arrest was induced by crossing heterozygous emi1 wt/mut zebrafish to generate homozygous emi1 mut/mut embryos referred as hi2648 in the manuscript. Homozygous mutants with cell cycle arrest were screened at 24 hpf as they have very clear phenotype by this time – these embryos are smaller and have bent tails and the heterozygous mutants and wildtype are indistinguishable. Zebrafish embryos were dechorionated using 1mg/mL Pronase Sigma P5147 1G for 5 7 minutes followed by washing in egg water. Embryos were dissociated as previously described in Wagner et. al. Science 2018. Briefly embryos were dissociated in 0.5mL LoBind microcentrifuge tubes that had been precoated with 10% w/v BSA for about 15 minutes at room temperature. They were homogenized in 1XDPBS/1% w/v BSA for 6 hpf to 10 hpf embryos early time points and in 500 µL FACSmax cell dissociation solution Genlantis T200100 for 14 hpf to 24 hpf embryos late time points. Cells were then filtered through a 40µm cell strainer mesh Fisher 352340 and centrifuged in a swinging bucket rotor at 200g for 1 minute early time points or 300g for 5 minutes late time points. Cells were then washed in 1XDPBS/1%BSA using the same centrifuge settings. They were then resuspended in 1XDPBS/0.5%BSA/18% optiprep density medium Sigma D1556 250ML. Cell concentration was manually quantified using hemocytometer and adjusted to a final concentration of 100 000 cells/mL before encapsulation. Library construction as detailed in Zilionis R. et al. 2016 Nature Protocols. Single cell barcoding and sequencing was done using droplet microfluidics also described in the same paper. AB wild type strains were used for all HUA perturbation experiments. Zebrafish mutant line hi2648 a mutant for the gene emi1 was kindly gifted by Dr. Jennifer Rhodes. Embryos were developed within the ambient temperature of Zebrafish development. Time of fertilization at 28.5 C was used to stage each clutch and this was confirmed using morphological features of the embryos. All zebrafish were housed in a facility overseen by the Harvard Medical Area Standing Committee on Animals our IACUC which performs regular inspections and under which we have an approved protocol for all animal procedures. tissue:whole embryo|treatment:control and emi1 homozygous mutants GSM8327219 GSM8327219: Perturbation experiment 3 24 hpf biological replicate 1 to 3 technical replicate 1; Danio rerio; OTHER GSM8327219 r1 GSM8327219 1 Zebrafish embryos were dechorionated using 1mg/mL Pronase Sigma P5147 1G for 5 7 minutes followed by washing in egg water. Embryos were dissociated as previously described in Wagner et. al. Science 2018. Briefly embryos were dissociated in 0.5mL LoBind microcentrifuge tubes that had been precoated with 10% w/v BSA for about 15 minutes at room temperature. They were homogenized in 1XDPBS/1% w/v BSA for 6 hpf to 10 hpf embryos early time points and in 500 µL FACSmax cell dissociation solution Genlantis T200100 for 14 hpf to 24 hpf embryos late time points. Cells were then filtered through a 40µm cell strainer mesh Fisher 352340 and centrifuged in a swinging bucket rotor at 200g for 1 minute early time points or 300g for 5 minutes late time points. Cells were then washed in 1XDPBS/1%BSA using the same centrifuge settings. They were then resuspended in 1XDPBS/0.5%BSA/18% optiprep density medium Sigma D1556 250ML. Cell concentration was manually quantified using hemocytometer and adjusted to a final concentration of 100 000 cells/mL before encapsulation. Library construction as detailed in Zilionis R. et al. 2016 Nature Protocols. Single cell barcoding and sequencing was done using droplet microfluidics also described in the same paper.   OTHER TRANSCRIPTOMIC other PAIRED ILLUMINA NextSeq 500   SRP513754     pert_exp3_brep1_2_3_trep1_S0_L001_R1_001.fastq.gz pert_exp3_brep1_2_3_trep1_S0_L001_R2_001.fastq.gz pert_exp3_brep1_2_3_trep1_S0_L001_R3_001.fastq.gz pert_exp3_brep1_2_3_trep1_S0_L001_R4_001.fastq.gz fastq fastq fastq fastq 11110219029.0 122090319.0 GSM8327219 r1 0:61 1:8 2:8 3:14 A:2089393711;C:1503585110;G:1499880828;T:2353645774;N:1004036 61 8 8 14 2089393711 1503585110 1499880828 2353645774 1004036 SRX24912670 SRS21618604 SRA1898111 Harvard University Harvard University                       B   usable mapping rate illumina nextseq unknown other trueseq sc single_cell_droplet indrops   United States 2024-06-13 Multi-stage Embryo Whole Organism All anatomical structures
32730 32730 SRR29398869 SRX24912670 SRS21618604 SRP513754 PRJNA1123686 Cell state transitions are decoupled from cell division during early embryo development [I] GSE269784 Other As tissues develop cells divide and differentiate concurrently. Conflicting evidence shows that cell division is either dispensable or required for formation of cell types. To determine the role of cell division in differentiation we arrested the cell cycle in zebrafish embryos using two independent approaches and profiled them at single cell resolution. We show that cell division is dispensable for differentiation of all embryonic tissues during initial cell type differentiation from early gastrulation to the end of segmentation. However in the absence of cell division differentiation slows down in some cell types and cells exhibit global stress responses. While differentiation is robust to blocking cell division the proportions of cells across cell states are not but show evidence of partial compensation. This work clarifies our understanding of the role of cell division in development and showcases the utility of combining embryo wide perturbations with single cell RNA sequencing to uncover the role of common biological processes across multiple tissues. Overall design: We arrested the cell cycle in zebrafish embryos at 6 hpf using two independent approaches: a chemical approach hydroxyurea and aphidicolin HUA and a genetic approach involving a loss of function mutation in the gene emi1 allele hi2648. We tracked differentiation dynamics using single cell RNA sequencing scRNA seq on embryos spanning 6–24 hpf for HUA treated and control embryos and at 24 hpf for emi1 mutant embryos. Overall we have collected multiple replicates for control embryos ABs at 6 8 10 14 18 21 hpf and 24 hpf for HUA treated at 8 10 14 hpf and 24 hpf and for emi1 mutants at 24 hpf. Details about the samples can be found in the supplementary file "sample information.txt"   pubmed:37546736   Perturbation experiment 3 24 hpf biological replicate 1 to 3 technical replicate 1 GSM8327219   source name:whole embryo|tissue:whole embryo|treatment:control and emi1 homozygous mutants|geo loc name:missing|collection date:missing Perturbation experiment 3 24 hpf biological replicate 1 to 3 technical replicate 1 Multiple samples are pooled for each sequencing run. Raw FASTQ were prepared from Illumina bcl files in a format compatible with the inDrops.py pipeline. Each nextseq run results in 16 FASTQ files 4 lanes x 4 reads per lane. For some pooled libraries sequencing was run twice to get more UMIs per cell and hence we have deposited 16x2 = 32 raw files for those sequencing samples. The illumina library indices of different samples in a run are provided in the meta data. These can be used to demultiplex the raw file into separate libraries. The read files from an inDrops run have the following content: * R1 001.fastq.gz : contains the three prime UTR cDNA read * R2 001.fastq.gz : contains the first half of the cell barcode * R3 001.fastq.gz : contains the library index for demultiplexing libraries * R4 001.fastq.gz : contains the second half of the barcode and the UMI. All subsequent processing steps from FASTQ files to count matrixes were performed using the custom pipeline for inDrops data analysis github.com/indrops. Single cell transcriptomes were barcoded using inDrops Klein et al Cell 2015. Standard transcriptome RNA seq libraries were processed as reported in Zilionis et al. Nature Protocol 2016 using inDrops v3 protocol. The transcriptome libraries were sequenced on reads Illumina NextSeq 500. Libraries used standard Illumina sequencing primers and 61 cycles for Read1 14 cycles for Read2 8 cycles each for IndexRead1 and IndexRead2. Raw fastq files was processed using inDrops.py pipeline github.com/indrops/indrops. Sequenced reads were mapped to a zebrafish reference transcriptome built from the zebrafish GRCz10 genome assembly Assembly Accession: GCF 000002035.5 using bowtie version 1.1.143. To obtain the final counts matrix used for data analysis total count filters were applied as described in Kukreja et. al. 2024 method section "Single cell RNA seq Data preprocessing" Assembly: GRCz10 genome assembly Assembly Accession: GCF 000002035.5 Supplementary files format and content: Raw counts tsv.gz: cell ba… whole embryo Zebrafish embryos were arrested for cell cycle using two complementary approaches. S phase cell cycle arrest was induced using a cocktail of drugs – hydroxyurea Sigma Aldrich H8627 5G at 20 mM and aphidicolin Sigma Aldrich A0781 5MG at 150 µM concentration in 1% dimethyl sulfoxide DMSO in egg water. G2/M phase arrest was induced by crossing heterozygous emi1 wt/mut zebrafish to generate homozygous emi1 mut/mut embryos referred as hi2648 in the manuscript. Homozygous mutants with cell cycle arrest were screened at 24 hpf as they have very clear phenotype by this time – these embryos are smaller and have bent tails and the heterozygous mutants and wildtype are indistinguishable. Zebrafish embryos were dechorionated using 1mg/mL Pronase Sigma P5147 1G for 5 7 minutes followed by washing in egg water. Embryos were dissociated as previously described in Wagner et. al. Science 2018. Briefly embryos were dissociated in 0.5mL LoBind microcentrifuge tubes that had been precoated with 10% w/v BSA for about 15 minutes at room temperature. They were homogenized in 1XDPBS/1% w/v BSA for 6 hpf to 10 hpf embryos early time points and in 500 µL FACSmax cell dissociation solution Genlantis T200100 for 14 hpf to 24 hpf embryos late time points. Cells were then filtered through a 40µm cell strainer mesh Fisher 352340 and centrifuged in a swinging bucket rotor at 200g for 1 minute early time points or 300g for 5 minutes late time points. Cells were then washed in 1XDPBS/1%BSA using the same centrifuge settings. They were then resuspended in 1XDPBS/0.5%BSA/18% optiprep density medium Sigma D1556 250ML. Cell concentration was manually quantified using hemocytometer and adjusted to a final concentration of 100 000 cells/mL before encapsulation. Library construction as detailed in Zilionis R. et al. 2016 Nature Protocols. Single cell barcoding and sequencing was done using droplet microfluidics also described in the same paper. AB wild type strains were used for all HUA perturbation experiments. Zebrafish mutant line hi2648 a mutant for the gene emi1 was kindly gifted by Dr. Jennifer Rhodes. Embryos were developed within the ambient temperature of Zebrafish development. Time of fertilization at 28.5 C was used to stage each clutch and this was confirmed using morphological features of the embryos. All zebrafish were housed in a facility overseen by the Harvard Medical Area Standing Committee on Animals our IACUC which performs regular inspections and under which we have an approved protocol for all animal procedures. tissue:whole embryo|treatment:control and emi1 homozygous mutants GSM8327219 GSM8327219: Perturbation experiment 3 24 hpf biological replicate 1 to 3 technical replicate 1; Danio rerio; OTHER GSM8327219 r1 GSM8327219 1 Zebrafish embryos were dechorionated using 1mg/mL Pronase Sigma P5147 1G for 5 7 minutes followed by washing in egg water. Embryos were dissociated as previously described in Wagner et. al. Science 2018. Briefly embryos were dissociated in 0.5mL LoBind microcentrifuge tubes that had been precoated with 10% w/v BSA for about 15 minutes at room temperature. They were homogenized in 1XDPBS/1% w/v BSA for 6 hpf to 10 hpf embryos early time points and in 500 µL FACSmax cell dissociation solution Genlantis T200100 for 14 hpf to 24 hpf embryos late time points. Cells were then filtered through a 40µm cell strainer mesh Fisher 352340 and centrifuged in a swinging bucket rotor at 200g for 1 minute early time points or 300g for 5 minutes late time points. Cells were then washed in 1XDPBS/1%BSA using the same centrifuge settings. They were then resuspended in 1XDPBS/0.5%BSA/18% optiprep density medium Sigma D1556 250ML. Cell concentration was manually quantified using hemocytometer and adjusted to a final concentration of 100 000 cells/mL before encapsulation. Library construction as detailed in Zilionis R. et al. 2016 Nature Protocols. Single cell barcoding and sequencing was done using droplet microfluidics also described in the same paper.   OTHER TRANSCRIPTOMIC other PAIRED ILLUMINA NextSeq 500   SRP513754     pert_exp3_brep1_2_3_trep1_S0_L002_R1_001.fastq.gz pert_exp3_brep1_2_3_trep1_S0_L002_R2_001.fastq.gz pert_exp3_brep1_2_3_trep1_S0_L002_R3_001.fastq.gz pert_exp3_brep1_2_3_trep1_S0_L002_R4_001.fastq.gz fastq fastq fastq fastq 10960533857.0 120445427.0 GSM8327219 r2 0:61 1:8 2:8 3:14 A:2060454932;C:1484110501;G:1481072325;T:2320147865;N:1385424 61 8 8 14 2060454932 1484110501 1481072325 2320147865 1385424 SRX24912670 SRS21618604 SRA1898111 Harvard University Harvard University                       B   usable mapping rate illumina nextseq unknown other trueseq sc single_cell_droplet indrops   United States 2024-06-13 Multi-stage Embryo Whole Organism All anatomical structures
32731 32731 SRR29398870 SRX24912670 SRS21618604 SRP513754 PRJNA1123686 Cell state transitions are decoupled from cell division during early embryo development [I] GSE269784 Other As tissues develop cells divide and differentiate concurrently. Conflicting evidence shows that cell division is either dispensable or required for formation of cell types. To determine the role of cell division in differentiation we arrested the cell cycle in zebrafish embryos using two independent approaches and profiled them at single cell resolution. We show that cell division is dispensable for differentiation of all embryonic tissues during initial cell type differentiation from early gastrulation to the end of segmentation. However in the absence of cell division differentiation slows down in some cell types and cells exhibit global stress responses. While differentiation is robust to blocking cell division the proportions of cells across cell states are not but show evidence of partial compensation. This work clarifies our understanding of the role of cell division in development and showcases the utility of combining embryo wide perturbations with single cell RNA sequencing to uncover the role of common biological processes across multiple tissues. Overall design: We arrested the cell cycle in zebrafish embryos at 6 hpf using two independent approaches: a chemical approach hydroxyurea and aphidicolin HUA and a genetic approach involving a loss of function mutation in the gene emi1 allele hi2648. We tracked differentiation dynamics using single cell RNA sequencing scRNA seq on embryos spanning 6–24 hpf for HUA treated and control embryos and at 24 hpf for emi1 mutant embryos. Overall we have collected multiple replicates for control embryos ABs at 6 8 10 14 18 21 hpf and 24 hpf for HUA treated at 8 10 14 hpf and 24 hpf and for emi1 mutants at 24 hpf. Details about the samples can be found in the supplementary file "sample information.txt"   pubmed:37546736   Perturbation experiment 3 24 hpf biological replicate 1 to 3 technical replicate 1 GSM8327219   source name:whole embryo|tissue:whole embryo|treatment:control and emi1 homozygous mutants|geo loc name:missing|collection date:missing Perturbation experiment 3 24 hpf biological replicate 1 to 3 technical replicate 1 Multiple samples are pooled for each sequencing run. Raw FASTQ were prepared from Illumina bcl files in a format compatible with the inDrops.py pipeline. Each nextseq run results in 16 FASTQ files 4 lanes x 4 reads per lane. For some pooled libraries sequencing was run twice to get more UMIs per cell and hence we have deposited 16x2 = 32 raw files for those sequencing samples. The illumina library indices of different samples in a run are provided in the meta data. These can be used to demultiplex the raw file into separate libraries. The read files from an inDrops run have the following content: * R1 001.fastq.gz : contains the three prime UTR cDNA read * R2 001.fastq.gz : contains the first half of the cell barcode * R3 001.fastq.gz : contains the library index for demultiplexing libraries * R4 001.fastq.gz : contains the second half of the barcode and the UMI. All subsequent processing steps from FASTQ files to count matrixes were performed using the custom pipeline for inDrops data analysis github.com/indrops. Single cell transcriptomes were barcoded using inDrops Klein et al Cell 2015. Standard transcriptome RNA seq libraries were processed as reported in Zilionis et al. Nature Protocol 2016 using inDrops v3 protocol. The transcriptome libraries were sequenced on reads Illumina NextSeq 500. Libraries used standard Illumina sequencing primers and 61 cycles for Read1 14 cycles for Read2 8 cycles each for IndexRead1 and IndexRead2. Raw fastq files was processed using inDrops.py pipeline github.com/indrops/indrops. Sequenced reads were mapped to a zebrafish reference transcriptome built from the zebrafish GRCz10 genome assembly Assembly Accession: GCF 000002035.5 using bowtie version 1.1.143. To obtain the final counts matrix used for data analysis total count filters were applied as described in Kukreja et. al. 2024 method section "Single cell RNA seq Data preprocessing" Assembly: GRCz10 genome assembly Assembly Accession: GCF 000002035.5 Supplementary files format and content: Raw counts tsv.gz: cell ba… whole embryo Zebrafish embryos were arrested for cell cycle using two complementary approaches. S phase cell cycle arrest was induced using a cocktail of drugs – hydroxyurea Sigma Aldrich H8627 5G at 20 mM and aphidicolin Sigma Aldrich A0781 5MG at 150 µM concentration in 1% dimethyl sulfoxide DMSO in egg water. G2/M phase arrest was induced by crossing heterozygous emi1 wt/mut zebrafish to generate homozygous emi1 mut/mut embryos referred as hi2648 in the manuscript. Homozygous mutants with cell cycle arrest were screened at 24 hpf as they have very clear phenotype by this time – these embryos are smaller and have bent tails and the heterozygous mutants and wildtype are indistinguishable. Zebrafish embryos were dechorionated using 1mg/mL Pronase Sigma P5147 1G for 5 7 minutes followed by washing in egg water. Embryos were dissociated as previously described in Wagner et. al. Science 2018. Briefly embryos were dissociated in 0.5mL LoBind microcentrifuge tubes that had been precoated with 10% w/v BSA for about 15 minutes at room temperature. They were homogenized in 1XDPBS/1% w/v BSA for 6 hpf to 10 hpf embryos early time points and in 500 µL FACSmax cell dissociation solution Genlantis T200100 for 14 hpf to 24 hpf embryos late time points. Cells were then filtered through a 40µm cell strainer mesh Fisher 352340 and centrifuged in a swinging bucket rotor at 200g for 1 minute early time points or 300g for 5 minutes late time points. Cells were then washed in 1XDPBS/1%BSA using the same centrifuge settings. They were then resuspended in 1XDPBS/0.5%BSA/18% optiprep density medium Sigma D1556 250ML. Cell concentration was manually quantified using hemocytometer and adjusted to a final concentration of 100 000 cells/mL before encapsulation. Library construction as detailed in Zilionis R. et al. 2016 Nature Protocols. Single cell barcoding and sequencing was done using droplet microfluidics also described in the same paper. AB wild type strains were used for all HUA perturbation experiments. Zebrafish mutant line hi2648 a mutant for the gene emi1 was kindly gifted by Dr. Jennifer Rhodes. Embryos were developed within the ambient temperature of Zebrafish development. Time of fertilization at 28.5 C was used to stage each clutch and this was confirmed using morphological features of the embryos. All zebrafish were housed in a facility overseen by the Harvard Medical Area Standing Committee on Animals our IACUC which performs regular inspections and under which we have an approved protocol for all animal procedures. tissue:whole embryo|treatment:control and emi1 homozygous mutants GSM8327219 GSM8327219: Perturbation experiment 3 24 hpf biological replicate 1 to 3 technical replicate 1; Danio rerio; OTHER GSM8327219 r1 GSM8327219 1 Zebrafish embryos were dechorionated using 1mg/mL Pronase Sigma P5147 1G for 5 7 minutes followed by washing in egg water. Embryos were dissociated as previously described in Wagner et. al. Science 2018. Briefly embryos were dissociated in 0.5mL LoBind microcentrifuge tubes that had been precoated with 10% w/v BSA for about 15 minutes at room temperature. They were homogenized in 1XDPBS/1% w/v BSA for 6 hpf to 10 hpf embryos early time points and in 500 µL FACSmax cell dissociation solution Genlantis T200100 for 14 hpf to 24 hpf embryos late time points. Cells were then filtered through a 40µm cell strainer mesh Fisher 352340 and centrifuged in a swinging bucket rotor at 200g for 1 minute early time points or 300g for 5 minutes late time points. Cells were then washed in 1XDPBS/1%BSA using the same centrifuge settings. They were then resuspended in 1XDPBS/0.5%BSA/18% optiprep density medium Sigma D1556 250ML. Cell concentration was manually quantified using hemocytometer and adjusted to a final concentration of 100 000 cells/mL before encapsulation. Library construction as detailed in Zilionis R. et al. 2016 Nature Protocols. Single cell barcoding and sequencing was done using droplet microfluidics also described in the same paper.   OTHER TRANSCRIPTOMIC other PAIRED ILLUMINA NextSeq 500   SRP513754     pert_exp3_brep1_2_3_trep1_S0_L003_R1_001.fastq.gz pert_exp3_brep1_2_3_trep1_S0_L003_R2_001.fastq.gz pert_exp3_brep1_2_3_trep1_S0_L003_R3_001.fastq.gz pert_exp3_brep1_2_3_trep1_S0_L003_R4_001.fastq.gz fastq fastq fastq fastq 11164280309.0 122684399.0 GSM8327219 r3 0:61 1:8 2:8 3:14 A:2098548238;C:1513159748;G:1508645874;T:2362720749;N:673730 61 8 8 14 2098548238 1513159748 1508645874 2362720749 673730 SRX24912670 SRS21618604 SRA1898111 Harvard University Harvard University                       B   usable mapping rate illumina nextseq unknown other trueseq sc single_cell_droplet indrops   United States 2024-06-13 Multi-stage Embryo Whole Organism All anatomical structures
32732 32732 SRR29398871 SRX24912670 SRS21618604 SRP513754 PRJNA1123686 Cell state transitions are decoupled from cell division during early embryo development [I] GSE269784 Other As tissues develop cells divide and differentiate concurrently. Conflicting evidence shows that cell division is either dispensable or required for formation of cell types. To determine the role of cell division in differentiation we arrested the cell cycle in zebrafish embryos using two independent approaches and profiled them at single cell resolution. We show that cell division is dispensable for differentiation of all embryonic tissues during initial cell type differentiation from early gastrulation to the end of segmentation. However in the absence of cell division differentiation slows down in some cell types and cells exhibit global stress responses. While differentiation is robust to blocking cell division the proportions of cells across cell states are not but show evidence of partial compensation. This work clarifies our understanding of the role of cell division in development and showcases the utility of combining embryo wide perturbations with single cell RNA sequencing to uncover the role of common biological processes across multiple tissues. Overall design: We arrested the cell cycle in zebrafish embryos at 6 hpf using two independent approaches: a chemical approach hydroxyurea and aphidicolin HUA and a genetic approach involving a loss of function mutation in the gene emi1 allele hi2648. We tracked differentiation dynamics using single cell RNA sequencing scRNA seq on embryos spanning 6–24 hpf for HUA treated and control embryos and at 24 hpf for emi1 mutant embryos. Overall we have collected multiple replicates for control embryos ABs at 6 8 10 14 18 21 hpf and 24 hpf for HUA treated at 8 10 14 hpf and 24 hpf and for emi1 mutants at 24 hpf. Details about the samples can be found in the supplementary file "sample information.txt"   pubmed:37546736   Perturbation experiment 3 24 hpf biological replicate 1 to 3 technical replicate 1 GSM8327219   source name:whole embryo|tissue:whole embryo|treatment:control and emi1 homozygous mutants|geo loc name:missing|collection date:missing Perturbation experiment 3 24 hpf biological replicate 1 to 3 technical replicate 1 Multiple samples are pooled for each sequencing run. Raw FASTQ were prepared from Illumina bcl files in a format compatible with the inDrops.py pipeline. Each nextseq run results in 16 FASTQ files 4 lanes x 4 reads per lane. For some pooled libraries sequencing was run twice to get more UMIs per cell and hence we have deposited 16x2 = 32 raw files for those sequencing samples. The illumina library indices of different samples in a run are provided in the meta data. These can be used to demultiplex the raw file into separate libraries. The read files from an inDrops run have the following content: * R1 001.fastq.gz : contains the three prime UTR cDNA read * R2 001.fastq.gz : contains the first half of the cell barcode * R3 001.fastq.gz : contains the library index for demultiplexing libraries * R4 001.fastq.gz : contains the second half of the barcode and the UMI. All subsequent processing steps from FASTQ files to count matrixes were performed using the custom pipeline for inDrops data analysis github.com/indrops. Single cell transcriptomes were barcoded using inDrops Klein et al Cell 2015. Standard transcriptome RNA seq libraries were processed as reported in Zilionis et al. Nature Protocol 2016 using inDrops v3 protocol. The transcriptome libraries were sequenced on reads Illumina NextSeq 500. Libraries used standard Illumina sequencing primers and 61 cycles for Read1 14 cycles for Read2 8 cycles each for IndexRead1 and IndexRead2. Raw fastq files was processed using inDrops.py pipeline github.com/indrops/indrops. Sequenced reads were mapped to a zebrafish reference transcriptome built from the zebrafish GRCz10 genome assembly Assembly Accession: GCF 000002035.5 using bowtie version 1.1.143. To obtain the final counts matrix used for data analysis total count filters were applied as described in Kukreja et. al. 2024 method section "Single cell RNA seq Data preprocessing" Assembly: GRCz10 genome assembly Assembly Accession: GCF 000002035.5 Supplementary files format and content: Raw counts tsv.gz: cell ba… whole embryo Zebrafish embryos were arrested for cell cycle using two complementary approaches. S phase cell cycle arrest was induced using a cocktail of drugs – hydroxyurea Sigma Aldrich H8627 5G at 20 mM and aphidicolin Sigma Aldrich A0781 5MG at 150 µM concentration in 1% dimethyl sulfoxide DMSO in egg water. G2/M phase arrest was induced by crossing heterozygous emi1 wt/mut zebrafish to generate homozygous emi1 mut/mut embryos referred as hi2648 in the manuscript. Homozygous mutants with cell cycle arrest were screened at 24 hpf as they have very clear phenotype by this time – these embryos are smaller and have bent tails and the heterozygous mutants and wildtype are indistinguishable. Zebrafish embryos were dechorionated using 1mg/mL Pronase Sigma P5147 1G for 5 7 minutes followed by washing in egg water. Embryos were dissociated as previously described in Wagner et. al. Science 2018. Briefly embryos were dissociated in 0.5mL LoBind microcentrifuge tubes that had been precoated with 10% w/v BSA for about 15 minutes at room temperature. They were homogenized in 1XDPBS/1% w/v BSA for 6 hpf to 10 hpf embryos early time points and in 500 µL FACSmax cell dissociation solution Genlantis T200100 for 14 hpf to 24 hpf embryos late time points. Cells were then filtered through a 40µm cell strainer mesh Fisher 352340 and centrifuged in a swinging bucket rotor at 200g for 1 minute early time points or 300g for 5 minutes late time points. Cells were then washed in 1XDPBS/1%BSA using the same centrifuge settings. They were then resuspended in 1XDPBS/0.5%BSA/18% optiprep density medium Sigma D1556 250ML. Cell concentration was manually quantified using hemocytometer and adjusted to a final concentration of 100 000 cells/mL before encapsulation. Library construction as detailed in Zilionis R. et al. 2016 Nature Protocols. Single cell barcoding and sequencing was done using droplet microfluidics also described in the same paper. AB wild type strains were used for all HUA perturbation experiments. Zebrafish mutant line hi2648 a mutant for the gene emi1 was kindly gifted by Dr. Jennifer Rhodes. Embryos were developed within the ambient temperature of Zebrafish development. Time of fertilization at 28.5 C was used to stage each clutch and this was confirmed using morphological features of the embryos. All zebrafish were housed in a facility overseen by the Harvard Medical Area Standing Committee on Animals our IACUC which performs regular inspections and under which we have an approved protocol for all animal procedures. tissue:whole embryo|treatment:control and emi1 homozygous mutants GSM8327219 GSM8327219: Perturbation experiment 3 24 hpf biological replicate 1 to 3 technical replicate 1; Danio rerio; OTHER GSM8327219 r1 GSM8327219 1 Zebrafish embryos were dechorionated using 1mg/mL Pronase Sigma P5147 1G for 5 7 minutes followed by washing in egg water. Embryos were dissociated as previously described in Wagner et. al. Science 2018. Briefly embryos were dissociated in 0.5mL LoBind microcentrifuge tubes that had been precoated with 10% w/v BSA for about 15 minutes at room temperature. They were homogenized in 1XDPBS/1% w/v BSA for 6 hpf to 10 hpf embryos early time points and in 500 µL FACSmax cell dissociation solution Genlantis T200100 for 14 hpf to 24 hpf embryos late time points. Cells were then filtered through a 40µm cell strainer mesh Fisher 352340 and centrifuged in a swinging bucket rotor at 200g for 1 minute early time points or 300g for 5 minutes late time points. Cells were then washed in 1XDPBS/1%BSA using the same centrifuge settings. They were then resuspended in 1XDPBS/0.5%BSA/18% optiprep density medium Sigma D1556 250ML. Cell concentration was manually quantified using hemocytometer and adjusted to a final concentration of 100 000 cells/mL before encapsulation. Library construction as detailed in Zilionis R. et al. 2016 Nature Protocols. Single cell barcoding and sequencing was done using droplet microfluidics also described in the same paper.   OTHER TRANSCRIPTOMIC other PAIRED ILLUMINA NextSeq 500   SRP513754     pert_exp3_brep1_2_3_trep1_S0_L004_R1_001.fastq.gz pert_exp3_brep1_2_3_trep1_S0_L004_R2_001.fastq.gz pert_exp3_brep1_2_3_trep1_S0_L004_R3_001.fastq.gz pert_exp3_brep1_2_3_trep1_S0_L004_R4_001.fastq.gz fastq fastq fastq fastq 11112051587.0 122110457.0 GSM8327219 r4 0:61 1:8 2:8 3:14 A:2088203494;C:1504533428;G:1503021684;T:2351948933;N:1030338 61 8 8 14 2088203494 1504533428 1503021684 2351948933 1030338 SRX24912670 SRS21618604 SRA1898111 Harvard University Harvard University                       B   usable mapping rate illumina nextseq unknown other trueseq sc single_cell_droplet indrops   United States 2024-06-13 Multi-stage Embryo Whole Organism All anatomical structures
32733 32733 SRR29398872 SRX24912669 SRS21618603 SRP513754 PRJNA1123686 Cell state transitions are decoupled from cell division during early embryo development [I] GSE269784 Other As tissues develop cells divide and differentiate concurrently. Conflicting evidence shows that cell division is either dispensable or required for formation of cell types. To determine the role of cell division in differentiation we arrested the cell cycle in zebrafish embryos using two independent approaches and profiled them at single cell resolution. We show that cell division is dispensable for differentiation of all embryonic tissues during initial cell type differentiation from early gastrulation to the end of segmentation. However in the absence of cell division differentiation slows down in some cell types and cells exhibit global stress responses. While differentiation is robust to blocking cell division the proportions of cells across cell states are not but show evidence of partial compensation. This work clarifies our understanding of the role of cell division in development and showcases the utility of combining embryo wide perturbations with single cell RNA sequencing to uncover the role of common biological processes across multiple tissues. Overall design: We arrested the cell cycle in zebrafish embryos at 6 hpf using two independent approaches: a chemical approach hydroxyurea and aphidicolin HUA and a genetic approach involving a loss of function mutation in the gene emi1 allele hi2648. We tracked differentiation dynamics using single cell RNA sequencing scRNA seq on embryos spanning 6–24 hpf for HUA treated and control embryos and at 24 hpf for emi1 mutant embryos. Overall we have collected multiple replicates for control embryos ABs at 6 8 10 14 18 21 hpf and 24 hpf for HUA treated at 8 10 14 hpf and 24 hpf and for emi1 mutants at 24 hpf. Details about the samples can be found in the supplementary file "sample information.txt"   pubmed:37546736   Perturbation experiment 2 24 hpf biological replicate 3 technical replicate 1 to 4 GSM8327218   source name:whole embryo|tissue:whole embryo|treatment:1percent DMSO control and cell cycle arrest at 6 hpf|geo loc name:missing|collection date:missing Perturbation experiment 2 24 hpf biological replicate 3 technical replicate 1 to 4 Multiple samples are pooled for each sequencing run. Raw FASTQ were prepared from Illumina bcl files in a format compatible with the inDrops.py pipeline. Each nextseq run results in 16 FASTQ files 4 lanes x 4 reads per lane. For some pooled libraries sequencing was run twice to get more UMIs per cell and hence we have deposited 16x2 = 32 raw files for those sequencing samples. The illumina library indices of different samples in a run are provided in the meta data. These can be used to demultiplex the raw file into separate libraries. The read files from an inDrops run have the following content: * R1 001.fastq.gz : contains the three prime UTR cDNA read * R2 001.fastq.gz : contains the first half of the cell barcode * R3 001.fastq.gz : contains the library index for demultiplexing libraries * R4 001.fastq.gz : contains the second half of the barcode and the UMI. All subsequent processing steps from FASTQ files to count matrixes were performed using the custom pipeline for inDrops data analysis github.com/indrops. Single cell transcriptomes were barcoded using inDrops Klein et al Cell 2015. Standard transcriptome RNA seq libraries were processed as reported in Zilionis et al. Nature Protocol 2016 using inDrops v3 protocol. The transcriptome libraries were sequenced on reads Illumina NextSeq 500. Libraries used standard Illumina sequencing primers and 61 cycles for Read1 14 cycles for Read2 8 cycles each for IndexRead1 and IndexRead2. Raw fastq files was processed using inDrops.py pipeline github.com/indrops/indrops. Sequenced reads were mapped to a zebrafish reference transcriptome built from the zebrafish GRCz10 genome assembly Assembly Accession: GCF 000002035.5 using bowtie version 1.1.143. To obtain the final counts matrix used for data analysis total count filters were applied as described in Kukreja et. al. 2024 method section "Single cell RNA seq Data preprocessing" Assembly: GRCz10 genome assembly Assembly Accession: GCF 000002035.5 Supplementary files format and content: Raw counts tsv.gz: cell ba… whole embryo Zebrafish embryos were arrested for cell cycle using two complementary approaches. S phase cell cycle arrest was induced using a cocktail of drugs – hydroxyurea Sigma Aldrich H8627 5G at 20 mM and aphidicolin Sigma Aldrich A0781 5MG at 150 µM concentration in 1% dimethyl sulfoxide DMSO in egg water. G2/M phase arrest was induced by crossing heterozygous emi1 wt/mut zebrafish to generate homozygous emi1 mut/mut embryos referred as hi2648 in the manuscript. Homozygous mutants with cell cycle arrest were screened at 24 hpf as they have very clear phenotype by this time – these embryos are smaller and have bent tails and the heterozygous mutants and wildtype are indistinguishable. Zebrafish embryos were dechorionated using 1mg/mL Pronase Sigma P5147 1G for 5 7 minutes followed by washing in egg water. Embryos were dissociated as previously described in Wagner et. al. Science 2018. Briefly embryos were dissociated in 0.5mL LoBind microcentrifuge tubes that had been precoated with 10% w/v BSA for about 15 minutes at room temperature. They were homogenized in 1XDPBS/1% w/v BSA for 6 hpf to 10 hpf embryos early time points and in 500 µL FACSmax cell dissociation solution Genlantis T200100 for 14 hpf to 24 hpf embryos late time points. Cells were then filtered through a 40µm cell strainer mesh Fisher 352340 and centrifuged in a swinging bucket rotor at 200g for 1 minute early time points or 300g for 5 minutes late time points. Cells were then washed in 1XDPBS/1%BSA using the same centrifuge settings. They were then resuspended in 1XDPBS/0.5%BSA/18% optiprep density medium Sigma D1556 250ML. Cell concentration was manually quantified using hemocytometer and adjusted to a final concentration of 100 000 cells/mL before encapsulation. Library construction as detailed in Zilionis R. et al. 2016 Nature Protocols. Single cell barcoding and sequencing was done using droplet microfluidics also described in the same paper. AB wild type strains were used for all HUA perturbation experiments. Zebrafish mutant line hi2648 a mutant for the gene emi1 was kindly gifted by Dr. Jennifer Rhodes. Embryos were developed within the ambient temperature of Zebrafish development. Time of fertilization at 28.5 C was used to stage each clutch and this was confirmed using morphological features of the embryos. All zebrafish were housed in a facility overseen by the Harvard Medical Area Standing Committee on Animals our IACUC which performs regular inspections and under which we have an approved protocol for all animal procedures. tissue:whole embryo|treatment:1percent DMSO control and cell cycle arrest at 6 hpf GSM8327218 GSM8327218: Perturbation experiment 2 24 hpf biological replicate 3 technical replicate 1 to 4; Danio rerio; OTHER GSM8327218 r1 GSM8327218 1 Zebrafish embryos were dechorionated using 1mg/mL Pronase Sigma P5147 1G for 5 7 minutes followed by washing in egg water. Embryos were dissociated as previously described in Wagner et. al. Science 2018. Briefly embryos were dissociated in 0.5mL LoBind microcentrifuge tubes that had been precoated with 10% w/v BSA for about 15 minutes at room temperature. They were homogenized in 1XDPBS/1% w/v BSA for 6 hpf to 10 hpf embryos early time points and in 500 µL FACSmax cell dissociation solution Genlantis T200100 for 14 hpf to 24 hpf embryos late time points. Cells were then filtered through a 40µm cell strainer mesh Fisher 352340 and centrifuged in a swinging bucket rotor at 200g for 1 minute early time points or 300g for 5 minutes late time points. Cells were then washed in 1XDPBS/1%BSA using the same centrifuge settings. They were then resuspended in 1XDPBS/0.5%BSA/18% optiprep density medium Sigma D1556 250ML. Cell concentration was manually quantified using hemocytometer and adjusted to a final concentration of 100 000 cells/mL before encapsulation. Library construction as detailed in Zilionis R. et al. 2016 Nature Protocols. Single cell barcoding and sequencing was done using droplet microfluidics also described in the same paper.   OTHER TRANSCRIPTOMIC other PAIRED ILLUMINA NextSeq 500   SRP513754     pert_exp2_brep3_trep1_to_4_S0_L001_R1_001.fastq.gz pert_exp2_brep3_trep1_to_4_S0_L001_R2_001.fastq.gz pert_exp2_brep3_trep1_to_4_S0_L001_R3_001.fastq.gz pert_exp2_brep3_trep1_to_4_S0_L001_R4_001.fastq.gz fastq fastq fastq fastq 10415939716.0 114460876.0 GSM8327218 r1 0:61 1:8 2:8 3:14 A:1937284334;C:1499506650;G:1405060643;T:2140026232;N:235577 61 8 8 14 1937284334 1499506650 1405060643 2140026232 235577 SRX24912669 SRS21618603 SRA1898111 Harvard University Harvard University                       B   usable mapping rate illumina nextseq unknown other trueseq sc single_cell_droplet indrops   United States 2024-06-13 Multi-stage Embryo Whole Organism All anatomical structures
32734 32734 SRR29398873 SRX24912669 SRS21618603 SRP513754 PRJNA1123686 Cell state transitions are decoupled from cell division during early embryo development [I] GSE269784 Other As tissues develop cells divide and differentiate concurrently. Conflicting evidence shows that cell division is either dispensable or required for formation of cell types. To determine the role of cell division in differentiation we arrested the cell cycle in zebrafish embryos using two independent approaches and profiled them at single cell resolution. We show that cell division is dispensable for differentiation of all embryonic tissues during initial cell type differentiation from early gastrulation to the end of segmentation. However in the absence of cell division differentiation slows down in some cell types and cells exhibit global stress responses. While differentiation is robust to blocking cell division the proportions of cells across cell states are not but show evidence of partial compensation. This work clarifies our understanding of the role of cell division in development and showcases the utility of combining embryo wide perturbations with single cell RNA sequencing to uncover the role of common biological processes across multiple tissues. Overall design: We arrested the cell cycle in zebrafish embryos at 6 hpf using two independent approaches: a chemical approach hydroxyurea and aphidicolin HUA and a genetic approach involving a loss of function mutation in the gene emi1 allele hi2648. We tracked differentiation dynamics using single cell RNA sequencing scRNA seq on embryos spanning 6–24 hpf for HUA treated and control embryos and at 24 hpf for emi1 mutant embryos. Overall we have collected multiple replicates for control embryos ABs at 6 8 10 14 18 21 hpf and 24 hpf for HUA treated at 8 10 14 hpf and 24 hpf and for emi1 mutants at 24 hpf. Details about the samples can be found in the supplementary file "sample information.txt"   pubmed:37546736   Perturbation experiment 2 24 hpf biological replicate 3 technical replicate 1 to 4 GSM8327218   source name:whole embryo|tissue:whole embryo|treatment:1percent DMSO control and cell cycle arrest at 6 hpf|geo loc name:missing|collection date:missing Perturbation experiment 2 24 hpf biological replicate 3 technical replicate 1 to 4 Multiple samples are pooled for each sequencing run. Raw FASTQ were prepared from Illumina bcl files in a format compatible with the inDrops.py pipeline. Each nextseq run results in 16 FASTQ files 4 lanes x 4 reads per lane. For some pooled libraries sequencing was run twice to get more UMIs per cell and hence we have deposited 16x2 = 32 raw files for those sequencing samples. The illumina library indices of different samples in a run are provided in the meta data. These can be used to demultiplex the raw file into separate libraries. The read files from an inDrops run have the following content: * R1 001.fastq.gz : contains the three prime UTR cDNA read * R2 001.fastq.gz : contains the first half of the cell barcode * R3 001.fastq.gz : contains the library index for demultiplexing libraries * R4 001.fastq.gz : contains the second half of the barcode and the UMI. All subsequent processing steps from FASTQ files to count matrixes were performed using the custom pipeline for inDrops data analysis github.com/indrops. Single cell transcriptomes were barcoded using inDrops Klein et al Cell 2015. Standard transcriptome RNA seq libraries were processed as reported in Zilionis et al. Nature Protocol 2016 using inDrops v3 protocol. The transcriptome libraries were sequenced on reads Illumina NextSeq 500. Libraries used standard Illumina sequencing primers and 61 cycles for Read1 14 cycles for Read2 8 cycles each for IndexRead1 and IndexRead2. Raw fastq files was processed using inDrops.py pipeline github.com/indrops/indrops. Sequenced reads were mapped to a zebrafish reference transcriptome built from the zebrafish GRCz10 genome assembly Assembly Accession: GCF 000002035.5 using bowtie version 1.1.143. To obtain the final counts matrix used for data analysis total count filters were applied as described in Kukreja et. al. 2024 method section "Single cell RNA seq Data preprocessing" Assembly: GRCz10 genome assembly Assembly Accession: GCF 000002035.5 Supplementary files format and content: Raw counts tsv.gz: cell ba… whole embryo Zebrafish embryos were arrested for cell cycle using two complementary approaches. S phase cell cycle arrest was induced using a cocktail of drugs – hydroxyurea Sigma Aldrich H8627 5G at 20 mM and aphidicolin Sigma Aldrich A0781 5MG at 150 µM concentration in 1% dimethyl sulfoxide DMSO in egg water. G2/M phase arrest was induced by crossing heterozygous emi1 wt/mut zebrafish to generate homozygous emi1 mut/mut embryos referred as hi2648 in the manuscript. Homozygous mutants with cell cycle arrest were screened at 24 hpf as they have very clear phenotype by this time – these embryos are smaller and have bent tails and the heterozygous mutants and wildtype are indistinguishable. Zebrafish embryos were dechorionated using 1mg/mL Pronase Sigma P5147 1G for 5 7 minutes followed by washing in egg water. Embryos were dissociated as previously described in Wagner et. al. Science 2018. Briefly embryos were dissociated in 0.5mL LoBind microcentrifuge tubes that had been precoated with 10% w/v BSA for about 15 minutes at room temperature. They were homogenized in 1XDPBS/1% w/v BSA for 6 hpf to 10 hpf embryos early time points and in 500 µL FACSmax cell dissociation solution Genlantis T200100 for 14 hpf to 24 hpf embryos late time points. Cells were then filtered through a 40µm cell strainer mesh Fisher 352340 and centrifuged in a swinging bucket rotor at 200g for 1 minute early time points or 300g for 5 minutes late time points. Cells were then washed in 1XDPBS/1%BSA using the same centrifuge settings. They were then resuspended in 1XDPBS/0.5%BSA/18% optiprep density medium Sigma D1556 250ML. Cell concentration was manually quantified using hemocytometer and adjusted to a final concentration of 100 000 cells/mL before encapsulation. Library construction as detailed in Zilionis R. et al. 2016 Nature Protocols. Single cell barcoding and sequencing was done using droplet microfluidics also described in the same paper. AB wild type strains were used for all HUA perturbation experiments. Zebrafish mutant line hi2648 a mutant for the gene emi1 was kindly gifted by Dr. Jennifer Rhodes. Embryos were developed within the ambient temperature of Zebrafish development. Time of fertilization at 28.5 C was used to stage each clutch and this was confirmed using morphological features of the embryos. All zebrafish were housed in a facility overseen by the Harvard Medical Area Standing Committee on Animals our IACUC which performs regular inspections and under which we have an approved protocol for all animal procedures. tissue:whole embryo|treatment:1percent DMSO control and cell cycle arrest at 6 hpf GSM8327218 GSM8327218: Perturbation experiment 2 24 hpf biological replicate 3 technical replicate 1 to 4; Danio rerio; OTHER GSM8327218 r1 GSM8327218 1 Zebrafish embryos were dechorionated using 1mg/mL Pronase Sigma P5147 1G for 5 7 minutes followed by washing in egg water. Embryos were dissociated as previously described in Wagner et. al. Science 2018. Briefly embryos were dissociated in 0.5mL LoBind microcentrifuge tubes that had been precoated with 10% w/v BSA for about 15 minutes at room temperature. They were homogenized in 1XDPBS/1% w/v BSA for 6 hpf to 10 hpf embryos early time points and in 500 µL FACSmax cell dissociation solution Genlantis T200100 for 14 hpf to 24 hpf embryos late time points. Cells were then filtered through a 40µm cell strainer mesh Fisher 352340 and centrifuged in a swinging bucket rotor at 200g for 1 minute early time points or 300g for 5 minutes late time points. Cells were then washed in 1XDPBS/1%BSA using the same centrifuge settings. They were then resuspended in 1XDPBS/0.5%BSA/18% optiprep density medium Sigma D1556 250ML. Cell concentration was manually quantified using hemocytometer and adjusted to a final concentration of 100 000 cells/mL before encapsulation. Library construction as detailed in Zilionis R. et al. 2016 Nature Protocols. Single cell barcoding and sequencing was done using droplet microfluidics also described in the same paper.   OTHER TRANSCRIPTOMIC other PAIRED ILLUMINA NextSeq 500   SRP513754     pert_exp2_brep3_trep1_to_4_S0_L002_R1_001.fastq.gz pert_exp2_brep3_trep1_to_4_S0_L002_R2_001.fastq.gz pert_exp2_brep3_trep1_to_4_S0_L002_R3_001.fastq.gz pert_exp2_brep3_trep1_to_4_S0_L002_R4_001.fastq.gz fastq fastq fastq fastq 10347954344.0 113713784.0 GSM8327218 r2 0:61 1:8 2:8 3:14 A:1929648265;C:1483960768;G:1389914714;T:2132824805;N:192272 61 8 8 14 1929648265 1483960768 1389914714 2132824805 192272 SRX24912669 SRS21618603 SRA1898111 Harvard University Harvard University                       B   usable mapping rate illumina nextseq unknown other trueseq sc single_cell_droplet indrops   United States 2024-06-13 Multi-stage Embryo Whole Organism All anatomical structures
32735 32735 SRR29398874 SRX24912669 SRS21618603 SRP513754 PRJNA1123686 Cell state transitions are decoupled from cell division during early embryo development [I] GSE269784 Other As tissues develop cells divide and differentiate concurrently. Conflicting evidence shows that cell division is either dispensable or required for formation of cell types. To determine the role of cell division in differentiation we arrested the cell cycle in zebrafish embryos using two independent approaches and profiled them at single cell resolution. We show that cell division is dispensable for differentiation of all embryonic tissues during initial cell type differentiation from early gastrulation to the end of segmentation. However in the absence of cell division differentiation slows down in some cell types and cells exhibit global stress responses. While differentiation is robust to blocking cell division the proportions of cells across cell states are not but show evidence of partial compensation. This work clarifies our understanding of the role of cell division in development and showcases the utility of combining embryo wide perturbations with single cell RNA sequencing to uncover the role of common biological processes across multiple tissues. Overall design: We arrested the cell cycle in zebrafish embryos at 6 hpf using two independent approaches: a chemical approach hydroxyurea and aphidicolin HUA and a genetic approach involving a loss of function mutation in the gene emi1 allele hi2648. We tracked differentiation dynamics using single cell RNA sequencing scRNA seq on embryos spanning 6–24 hpf for HUA treated and control embryos and at 24 hpf for emi1 mutant embryos. Overall we have collected multiple replicates for control embryos ABs at 6 8 10 14 18 21 hpf and 24 hpf for HUA treated at 8 10 14 hpf and 24 hpf and for emi1 mutants at 24 hpf. Details about the samples can be found in the supplementary file "sample information.txt"   pubmed:37546736   Perturbation experiment 2 24 hpf biological replicate 3 technical replicate 1 to 4 GSM8327218   source name:whole embryo|tissue:whole embryo|treatment:1percent DMSO control and cell cycle arrest at 6 hpf|geo loc name:missing|collection date:missing Perturbation experiment 2 24 hpf biological replicate 3 technical replicate 1 to 4 Multiple samples are pooled for each sequencing run. Raw FASTQ were prepared from Illumina bcl files in a format compatible with the inDrops.py pipeline. Each nextseq run results in 16 FASTQ files 4 lanes x 4 reads per lane. For some pooled libraries sequencing was run twice to get more UMIs per cell and hence we have deposited 16x2 = 32 raw files for those sequencing samples. The illumina library indices of different samples in a run are provided in the meta data. These can be used to demultiplex the raw file into separate libraries. The read files from an inDrops run have the following content: * R1 001.fastq.gz : contains the three prime UTR cDNA read * R2 001.fastq.gz : contains the first half of the cell barcode * R3 001.fastq.gz : contains the library index for demultiplexing libraries * R4 001.fastq.gz : contains the second half of the barcode and the UMI. All subsequent processing steps from FASTQ files to count matrixes were performed using the custom pipeline for inDrops data analysis github.com/indrops. Single cell transcriptomes were barcoded using inDrops Klein et al Cell 2015. Standard transcriptome RNA seq libraries were processed as reported in Zilionis et al. Nature Protocol 2016 using inDrops v3 protocol. The transcriptome libraries were sequenced on reads Illumina NextSeq 500. Libraries used standard Illumina sequencing primers and 61 cycles for Read1 14 cycles for Read2 8 cycles each for IndexRead1 and IndexRead2. Raw fastq files was processed using inDrops.py pipeline github.com/indrops/indrops. Sequenced reads were mapped to a zebrafish reference transcriptome built from the zebrafish GRCz10 genome assembly Assembly Accession: GCF 000002035.5 using bowtie version 1.1.143. To obtain the final counts matrix used for data analysis total count filters were applied as described in Kukreja et. al. 2024 method section "Single cell RNA seq Data preprocessing" Assembly: GRCz10 genome assembly Assembly Accession: GCF 000002035.5 Supplementary files format and content: Raw counts tsv.gz: cell ba… whole embryo Zebrafish embryos were arrested for cell cycle using two complementary approaches. S phase cell cycle arrest was induced using a cocktail of drugs – hydroxyurea Sigma Aldrich H8627 5G at 20 mM and aphidicolin Sigma Aldrich A0781 5MG at 150 µM concentration in 1% dimethyl sulfoxide DMSO in egg water. G2/M phase arrest was induced by crossing heterozygous emi1 wt/mut zebrafish to generate homozygous emi1 mut/mut embryos referred as hi2648 in the manuscript. Homozygous mutants with cell cycle arrest were screened at 24 hpf as they have very clear phenotype by this time – these embryos are smaller and have bent tails and the heterozygous mutants and wildtype are indistinguishable. Zebrafish embryos were dechorionated using 1mg/mL Pronase Sigma P5147 1G for 5 7 minutes followed by washing in egg water. Embryos were dissociated as previously described in Wagner et. al. Science 2018. Briefly embryos were dissociated in 0.5mL LoBind microcentrifuge tubes that had been precoated with 10% w/v BSA for about 15 minutes at room temperature. They were homogenized in 1XDPBS/1% w/v BSA for 6 hpf to 10 hpf embryos early time points and in 500 µL FACSmax cell dissociation solution Genlantis T200100 for 14 hpf to 24 hpf embryos late time points. Cells were then filtered through a 40µm cell strainer mesh Fisher 352340 and centrifuged in a swinging bucket rotor at 200g for 1 minute early time points or 300g for 5 minutes late time points. Cells were then washed in 1XDPBS/1%BSA using the same centrifuge settings. They were then resuspended in 1XDPBS/0.5%BSA/18% optiprep density medium Sigma D1556 250ML. Cell concentration was manually quantified using hemocytometer and adjusted to a final concentration of 100 000 cells/mL before encapsulation. Library construction as detailed in Zilionis R. et al. 2016 Nature Protocols. Single cell barcoding and sequencing was done using droplet microfluidics also described in the same paper. AB wild type strains were used for all HUA perturbation experiments. Zebrafish mutant line hi2648 a mutant for the gene emi1 was kindly gifted by Dr. Jennifer Rhodes. Embryos were developed within the ambient temperature of Zebrafish development. Time of fertilization at 28.5 C was used to stage each clutch and this was confirmed using morphological features of the embryos. All zebrafish were housed in a facility overseen by the Harvard Medical Area Standing Committee on Animals our IACUC which performs regular inspections and under which we have an approved protocol for all animal procedures. tissue:whole embryo|treatment:1percent DMSO control and cell cycle arrest at 6 hpf GSM8327218 GSM8327218: Perturbation experiment 2 24 hpf biological replicate 3 technical replicate 1 to 4; Danio rerio; OTHER GSM8327218 r1 GSM8327218 1 Zebrafish embryos were dechorionated using 1mg/mL Pronase Sigma P5147 1G for 5 7 minutes followed by washing in egg water. Embryos were dissociated as previously described in Wagner et. al. Science 2018. Briefly embryos were dissociated in 0.5mL LoBind microcentrifuge tubes that had been precoated with 10% w/v BSA for about 15 minutes at room temperature. They were homogenized in 1XDPBS/1% w/v BSA for 6 hpf to 10 hpf embryos early time points and in 500 µL FACSmax cell dissociation solution Genlantis T200100 for 14 hpf to 24 hpf embryos late time points. Cells were then filtered through a 40µm cell strainer mesh Fisher 352340 and centrifuged in a swinging bucket rotor at 200g for 1 minute early time points or 300g for 5 minutes late time points. Cells were then washed in 1XDPBS/1%BSA using the same centrifuge settings. They were then resuspended in 1XDPBS/0.5%BSA/18% optiprep density medium Sigma D1556 250ML. Cell concentration was manually quantified using hemocytometer and adjusted to a final concentration of 100 000 cells/mL before encapsulation. Library construction as detailed in Zilionis R. et al. 2016 Nature Protocols. Single cell barcoding and sequencing was done using droplet microfluidics also described in the same paper.   OTHER TRANSCRIPTOMIC other PAIRED ILLUMINA NextSeq 500   SRP513754     pert_exp2_brep3_trep1_to_4_S0_L003_R1_001.fastq.gz pert_exp2_brep3_trep1_to_4_S0_L003_R2_001.fastq.gz pert_exp2_brep3_trep1_to_4_S0_L003_R3_001.fastq.gz pert_exp2_brep3_trep1_to_4_S0_L003_R4_001.fastq.gz fastq fastq fastq fastq 10349635660.0 113732260.0 GSM8327218 r3 0:61 1:8 2:8 3:14 A:1930191842;C:1471248043;G:1399416562;T:2136531503;N:279910 61 8 8 14 1930191842 1471248043 1399416562 2136531503 279910 SRX24912669 SRS21618603 SRA1898111 Harvard University Harvard University                       B   usable mapping rate illumina nextseq unknown other trueseq sc single_cell_droplet indrops   United States 2024-06-13 Multi-stage Embryo Whole Organism All anatomical structures
32736 32736 SRR29398875 SRX24912669 SRS21618603 SRP513754 PRJNA1123686 Cell state transitions are decoupled from cell division during early embryo development [I] GSE269784 Other As tissues develop cells divide and differentiate concurrently. Conflicting evidence shows that cell division is either dispensable or required for formation of cell types. To determine the role of cell division in differentiation we arrested the cell cycle in zebrafish embryos using two independent approaches and profiled them at single cell resolution. We show that cell division is dispensable for differentiation of all embryonic tissues during initial cell type differentiation from early gastrulation to the end of segmentation. However in the absence of cell division differentiation slows down in some cell types and cells exhibit global stress responses. While differentiation is robust to blocking cell division the proportions of cells across cell states are not but show evidence of partial compensation. This work clarifies our understanding of the role of cell division in development and showcases the utility of combining embryo wide perturbations with single cell RNA sequencing to uncover the role of common biological processes across multiple tissues. Overall design: We arrested the cell cycle in zebrafish embryos at 6 hpf using two independent approaches: a chemical approach hydroxyurea and aphidicolin HUA and a genetic approach involving a loss of function mutation in the gene emi1 allele hi2648. We tracked differentiation dynamics using single cell RNA sequencing scRNA seq on embryos spanning 6–24 hpf for HUA treated and control embryos and at 24 hpf for emi1 mutant embryos. Overall we have collected multiple replicates for control embryos ABs at 6 8 10 14 18 21 hpf and 24 hpf for HUA treated at 8 10 14 hpf and 24 hpf and for emi1 mutants at 24 hpf. Details about the samples can be found in the supplementary file "sample information.txt"   pubmed:37546736   Perturbation experiment 2 24 hpf biological replicate 3 technical replicate 1 to 4 GSM8327218   source name:whole embryo|tissue:whole embryo|treatment:1percent DMSO control and cell cycle arrest at 6 hpf|geo loc name:missing|collection date:missing Perturbation experiment 2 24 hpf biological replicate 3 technical replicate 1 to 4 Multiple samples are pooled for each sequencing run. Raw FASTQ were prepared from Illumina bcl files in a format compatible with the inDrops.py pipeline. Each nextseq run results in 16 FASTQ files 4 lanes x 4 reads per lane. For some pooled libraries sequencing was run twice to get more UMIs per cell and hence we have deposited 16x2 = 32 raw files for those sequencing samples. The illumina library indices of different samples in a run are provided in the meta data. These can be used to demultiplex the raw file into separate libraries. The read files from an inDrops run have the following content: * R1 001.fastq.gz : contains the three prime UTR cDNA read * R2 001.fastq.gz : contains the first half of the cell barcode * R3 001.fastq.gz : contains the library index for demultiplexing libraries * R4 001.fastq.gz : contains the second half of the barcode and the UMI. All subsequent processing steps from FASTQ files to count matrixes were performed using the custom pipeline for inDrops data analysis github.com/indrops. Single cell transcriptomes were barcoded using inDrops Klein et al Cell 2015. Standard transcriptome RNA seq libraries were processed as reported in Zilionis et al. Nature Protocol 2016 using inDrops v3 protocol. The transcriptome libraries were sequenced on reads Illumina NextSeq 500. Libraries used standard Illumina sequencing primers and 61 cycles for Read1 14 cycles for Read2 8 cycles each for IndexRead1 and IndexRead2. Raw fastq files was processed using inDrops.py pipeline github.com/indrops/indrops. Sequenced reads were mapped to a zebrafish reference transcriptome built from the zebrafish GRCz10 genome assembly Assembly Accession: GCF 000002035.5 using bowtie version 1.1.143. To obtain the final counts matrix used for data analysis total count filters were applied as described in Kukreja et. al. 2024 method section "Single cell RNA seq Data preprocessing" Assembly: GRCz10 genome assembly Assembly Accession: GCF 000002035.5 Supplementary files format and content: Raw counts tsv.gz: cell ba… whole embryo Zebrafish embryos were arrested for cell cycle using two complementary approaches. S phase cell cycle arrest was induced using a cocktail of drugs – hydroxyurea Sigma Aldrich H8627 5G at 20 mM and aphidicolin Sigma Aldrich A0781 5MG at 150 µM concentration in 1% dimethyl sulfoxide DMSO in egg water. G2/M phase arrest was induced by crossing heterozygous emi1 wt/mut zebrafish to generate homozygous emi1 mut/mut embryos referred as hi2648 in the manuscript. Homozygous mutants with cell cycle arrest were screened at 24 hpf as they have very clear phenotype by this time – these embryos are smaller and have bent tails and the heterozygous mutants and wildtype are indistinguishable. Zebrafish embryos were dechorionated using 1mg/mL Pronase Sigma P5147 1G for 5 7 minutes followed by washing in egg water. Embryos were dissociated as previously described in Wagner et. al. Science 2018. Briefly embryos were dissociated in 0.5mL LoBind microcentrifuge tubes that had been precoated with 10% w/v BSA for about 15 minutes at room temperature. They were homogenized in 1XDPBS/1% w/v BSA for 6 hpf to 10 hpf embryos early time points and in 500 µL FACSmax cell dissociation solution Genlantis T200100 for 14 hpf to 24 hpf embryos late time points. Cells were then filtered through a 40µm cell strainer mesh Fisher 352340 and centrifuged in a swinging bucket rotor at 200g for 1 minute early time points or 300g for 5 minutes late time points. Cells were then washed in 1XDPBS/1%BSA using the same centrifuge settings. They were then resuspended in 1XDPBS/0.5%BSA/18% optiprep density medium Sigma D1556 250ML. Cell concentration was manually quantified using hemocytometer and adjusted to a final concentration of 100 000 cells/mL before encapsulation. Library construction as detailed in Zilionis R. et al. 2016 Nature Protocols. Single cell barcoding and sequencing was done using droplet microfluidics also described in the same paper. AB wild type strains were used for all HUA perturbation experiments. Zebrafish mutant line hi2648 a mutant for the gene emi1 was kindly gifted by Dr. Jennifer Rhodes. Embryos were developed within the ambient temperature of Zebrafish development. Time of fertilization at 28.5 C was used to stage each clutch and this was confirmed using morphological features of the embryos. All zebrafish were housed in a facility overseen by the Harvard Medical Area Standing Committee on Animals our IACUC which performs regular inspections and under which we have an approved protocol for all animal procedures. tissue:whole embryo|treatment:1percent DMSO control and cell cycle arrest at 6 hpf GSM8327218 GSM8327218: Perturbation experiment 2 24 hpf biological replicate 3 technical replicate 1 to 4; Danio rerio; OTHER GSM8327218 r1 GSM8327218 1 Zebrafish embryos were dechorionated using 1mg/mL Pronase Sigma P5147 1G for 5 7 minutes followed by washing in egg water. Embryos were dissociated as previously described in Wagner et. al. Science 2018. Briefly embryos were dissociated in 0.5mL LoBind microcentrifuge tubes that had been precoated with 10% w/v BSA for about 15 minutes at room temperature. They were homogenized in 1XDPBS/1% w/v BSA for 6 hpf to 10 hpf embryos early time points and in 500 µL FACSmax cell dissociation solution Genlantis T200100 for 14 hpf to 24 hpf embryos late time points. Cells were then filtered through a 40µm cell strainer mesh Fisher 352340 and centrifuged in a swinging bucket rotor at 200g for 1 minute early time points or 300g for 5 minutes late time points. Cells were then washed in 1XDPBS/1%BSA using the same centrifuge settings. They were then resuspended in 1XDPBS/0.5%BSA/18% optiprep density medium Sigma D1556 250ML. Cell concentration was manually quantified using hemocytometer and adjusted to a final concentration of 100 000 cells/mL before encapsulation. Library construction as detailed in Zilionis R. et al. 2016 Nature Protocols. Single cell barcoding and sequencing was done using droplet microfluidics also described in the same paper.   OTHER TRANSCRIPTOMIC other PAIRED ILLUMINA NextSeq 500   SRP513754     pert_exp2_brep3_trep1_to_4_S0_L004_R1_001.fastq.gz pert_exp2_brep3_trep1_to_4_S0_L004_R2_001.fastq.gz pert_exp2_brep3_trep1_to_4_S0_L004_R3_001.fastq.gz pert_exp2_brep3_trep1_to_4_S0_L004_R4_001.fastq.gz fastq fastq fastq fastq 10428481518.0 114598698.0 GSM8327218 r4 0:61 1:8 2:8 3:14 A:1947321872;C:1488626558;G:1403484811;T:2150802963;N:284374 61 8 8 14 1947321872 1488626558 1403484811 2150802963 284374 SRX24912669 SRS21618603 SRA1898111 Harvard University Harvard University                       B   usable mapping rate illumina nextseq unknown other trueseq sc single_cell_droplet indrops   United States 2024-06-13 Multi-stage Embryo Whole Organism All anatomical structures
32737 32737 SRR29398876 SRX24912668 SRS21618601 SRP513754 PRJNA1123686 Cell state transitions are decoupled from cell division during early embryo development [I] GSE269784 Other As tissues develop cells divide and differentiate concurrently. Conflicting evidence shows that cell division is either dispensable or required for formation of cell types. To determine the role of cell division in differentiation we arrested the cell cycle in zebrafish embryos using two independent approaches and profiled them at single cell resolution. We show that cell division is dispensable for differentiation of all embryonic tissues during initial cell type differentiation from early gastrulation to the end of segmentation. However in the absence of cell division differentiation slows down in some cell types and cells exhibit global stress responses. While differentiation is robust to blocking cell division the proportions of cells across cell states are not but show evidence of partial compensation. This work clarifies our understanding of the role of cell division in development and showcases the utility of combining embryo wide perturbations with single cell RNA sequencing to uncover the role of common biological processes across multiple tissues. Overall design: We arrested the cell cycle in zebrafish embryos at 6 hpf using two independent approaches: a chemical approach hydroxyurea and aphidicolin HUA and a genetic approach involving a loss of function mutation in the gene emi1 allele hi2648. We tracked differentiation dynamics using single cell RNA sequencing scRNA seq on embryos spanning 6–24 hpf for HUA treated and control embryos and at 24 hpf for emi1 mutant embryos. Overall we have collected multiple replicates for control embryos ABs at 6 8 10 14 18 21 hpf and 24 hpf for HUA treated at 8 10 14 hpf and 24 hpf and for emi1 mutants at 24 hpf. Details about the samples can be found in the supplementary file "sample information.txt"   pubmed:37546736   Perturbation experiment 2 24 hpf biological replicate 2 technical replicate 1 to 6 GSM8327217   source name:whole embryo|tissue:whole embryo|treatment:1percent DMSO control and cell cycle arrest at 6 hpf|geo loc name:missing|collection date:missing Perturbation experiment 2 24 hpf biological replicate 2 technical replicate 1 to 6 Multiple samples are pooled for each sequencing run. Raw FASTQ were prepared from Illumina bcl files in a format compatible with the inDrops.py pipeline. Each nextseq run results in 16 FASTQ files 4 lanes x 4 reads per lane. For some pooled libraries sequencing was run twice to get more UMIs per cell and hence we have deposited 16x2 = 32 raw files for those sequencing samples. The illumina library indices of different samples in a run are provided in the meta data. These can be used to demultiplex the raw file into separate libraries. The read files from an inDrops run have the following content: * R1 001.fastq.gz : contains the three prime UTR cDNA read * R2 001.fastq.gz : contains the first half of the cell barcode * R3 001.fastq.gz : contains the library index for demultiplexing libraries * R4 001.fastq.gz : contains the second half of the barcode and the UMI. All subsequent processing steps from FASTQ files to count matrixes were performed using the custom pipeline for inDrops data analysis github.com/indrops. Single cell transcriptomes were barcoded using inDrops Klein et al Cell 2015. Standard transcriptome RNA seq libraries were processed as reported in Zilionis et al. Nature Protocol 2016 using inDrops v3 protocol. The transcriptome libraries were sequenced on reads Illumina NextSeq 500. Libraries used standard Illumina sequencing primers and 61 cycles for Read1 14 cycles for Read2 8 cycles each for IndexRead1 and IndexRead2. Raw fastq files was processed using inDrops.py pipeline github.com/indrops/indrops. Sequenced reads were mapped to a zebrafish reference transcriptome built from the zebrafish GRCz10 genome assembly Assembly Accession: GCF 000002035.5 using bowtie version 1.1.143. To obtain the final counts matrix used for data analysis total count filters were applied as described in Kukreja et. al. 2024 method section "Single cell RNA seq Data preprocessing" Assembly: GRCz10 genome assembly Assembly Accession: GCF 000002035.5 Supplementary files format and content: Raw counts tsv.gz: cell ba… whole embryo Zebrafish embryos were arrested for cell cycle using two complementary approaches. S phase cell cycle arrest was induced using a cocktail of drugs – hydroxyurea Sigma Aldrich H8627 5G at 20 mM and aphidicolin Sigma Aldrich A0781 5MG at 150 µM concentration in 1% dimethyl sulfoxide DMSO in egg water. G2/M phase arrest was induced by crossing heterozygous emi1 wt/mut zebrafish to generate homozygous emi1 mut/mut embryos referred as hi2648 in the manuscript. Homozygous mutants with cell cycle arrest were screened at 24 hpf as they have very clear phenotype by this time – these embryos are smaller and have bent tails and the heterozygous mutants and wildtype are indistinguishable. Zebrafish embryos were dechorionated using 1mg/mL Pronase Sigma P5147 1G for 5 7 minutes followed by washing in egg water. Embryos were dissociated as previously described in Wagner et. al. Science 2018. Briefly embryos were dissociated in 0.5mL LoBind microcentrifuge tubes that had been precoated with 10% w/v BSA for about 15 minutes at room temperature. They were homogenized in 1XDPBS/1% w/v BSA for 6 hpf to 10 hpf embryos early time points and in 500 µL FACSmax cell dissociation solution Genlantis T200100 for 14 hpf to 24 hpf embryos late time points. Cells were then filtered through a 40µm cell strainer mesh Fisher 352340 and centrifuged in a swinging bucket rotor at 200g for 1 minute early time points or 300g for 5 minutes late time points. Cells were then washed in 1XDPBS/1%BSA using the same centrifuge settings. They were then resuspended in 1XDPBS/0.5%BSA/18% optiprep density medium Sigma D1556 250ML. Cell concentration was manually quantified using hemocytometer and adjusted to a final concentration of 100 000 cells/mL before encapsulation. Library construction as detailed in Zilionis R. et al. 2016 Nature Protocols. Single cell barcoding and sequencing was done using droplet microfluidics also described in the same paper. AB wild type strains were used for all HUA perturbation experiments. Zebrafish mutant line hi2648 a mutant for the gene emi1 was kindly gifted by Dr. Jennifer Rhodes. Embryos were developed within the ambient temperature of Zebrafish development. Time of fertilization at 28.5 C was used to stage each clutch and this was confirmed using morphological features of the embryos. All zebrafish were housed in a facility overseen by the Harvard Medical Area Standing Committee on Animals our IACUC which performs regular inspections and under which we have an approved protocol for all animal procedures. tissue:whole embryo|treatment:1percent DMSO control and cell cycle arrest at 6 hpf GSM8327217 GSM8327217: Perturbation experiment 2 24 hpf biological replicate 2 technical replicate 1 to 6; Danio rerio; OTHER GSM8327217 r1 GSM8327217 1 Zebrafish embryos were dechorionated using 1mg/mL Pronase Sigma P5147 1G for 5 7 minutes followed by washing in egg water. Embryos were dissociated as previously described in Wagner et. al. Science 2018. Briefly embryos were dissociated in 0.5mL LoBind microcentrifuge tubes that had been precoated with 10% w/v BSA for about 15 minutes at room temperature. They were homogenized in 1XDPBS/1% w/v BSA for 6 hpf to 10 hpf embryos early time points and in 500 µL FACSmax cell dissociation solution Genlantis T200100 for 14 hpf to 24 hpf embryos late time points. Cells were then filtered through a 40µm cell strainer mesh Fisher 352340 and centrifuged in a swinging bucket rotor at 200g for 1 minute early time points or 300g for 5 minutes late time points. Cells were then washed in 1XDPBS/1%BSA using the same centrifuge settings. They were then resuspended in 1XDPBS/0.5%BSA/18% optiprep density medium Sigma D1556 250ML. Cell concentration was manually quantified using hemocytometer and adjusted to a final concentration of 100 000 cells/mL before encapsulation. Library construction as detailed in Zilionis R. et al. 2016 Nature Protocols. Single cell barcoding and sequencing was done using droplet microfluidics also described in the same paper.   OTHER TRANSCRIPTOMIC other PAIRED ILLUMINA NextSeq 500   SRP513754     pert_exp2_brep2_trep1_to_6_S0_L001_R1_001.fastq.gz pert_exp2_brep2_trep1_to_6_S0_L001_R2_001.fastq.gz pert_exp2_brep2_trep1_to_6_S0_L001_R3_001.fastq.gz pert_exp2_brep2_trep1_to_6_S0_L001_R4_001.fastq.gz fastq fastq fastq fastq 9307639614.0 102281754.0 GSM8327217 r1 0:61 1:8 2:8 3:14 A:1721795009;C:1288169328;G:1244888947;T:1984236555;N:97155 61 8 8 14 1721795009 1288169328 1244888947 1984236555 97155 SRX24912668 SRS21618601 SRA1898111 Harvard University Harvard University                       B   usable mapping rate illumina nextseq unknown other trueseq sc single_cell_droplet indrops   United States 2024-06-13 Multi-stage Embryo Whole Organism All anatomical structures
32738 32738 SRR29398877 SRX24912668 SRS21618601 SRP513754 PRJNA1123686 Cell state transitions are decoupled from cell division during early embryo development [I] GSE269784 Other As tissues develop cells divide and differentiate concurrently. Conflicting evidence shows that cell division is either dispensable or required for formation of cell types. To determine the role of cell division in differentiation we arrested the cell cycle in zebrafish embryos using two independent approaches and profiled them at single cell resolution. We show that cell division is dispensable for differentiation of all embryonic tissues during initial cell type differentiation from early gastrulation to the end of segmentation. However in the absence of cell division differentiation slows down in some cell types and cells exhibit global stress responses. While differentiation is robust to blocking cell division the proportions of cells across cell states are not but show evidence of partial compensation. This work clarifies our understanding of the role of cell division in development and showcases the utility of combining embryo wide perturbations with single cell RNA sequencing to uncover the role of common biological processes across multiple tissues. Overall design: We arrested the cell cycle in zebrafish embryos at 6 hpf using two independent approaches: a chemical approach hydroxyurea and aphidicolin HUA and a genetic approach involving a loss of function mutation in the gene emi1 allele hi2648. We tracked differentiation dynamics using single cell RNA sequencing scRNA seq on embryos spanning 6–24 hpf for HUA treated and control embryos and at 24 hpf for emi1 mutant embryos. Overall we have collected multiple replicates for control embryos ABs at 6 8 10 14 18 21 hpf and 24 hpf for HUA treated at 8 10 14 hpf and 24 hpf and for emi1 mutants at 24 hpf. Details about the samples can be found in the supplementary file "sample information.txt"   pubmed:37546736   Perturbation experiment 2 24 hpf biological replicate 2 technical replicate 1 to 6 GSM8327217   source name:whole embryo|tissue:whole embryo|treatment:1percent DMSO control and cell cycle arrest at 6 hpf|geo loc name:missing|collection date:missing Perturbation experiment 2 24 hpf biological replicate 2 technical replicate 1 to 6 Multiple samples are pooled for each sequencing run. Raw FASTQ were prepared from Illumina bcl files in a format compatible with the inDrops.py pipeline. Each nextseq run results in 16 FASTQ files 4 lanes x 4 reads per lane. For some pooled libraries sequencing was run twice to get more UMIs per cell and hence we have deposited 16x2 = 32 raw files for those sequencing samples. The illumina library indices of different samples in a run are provided in the meta data. These can be used to demultiplex the raw file into separate libraries. The read files from an inDrops run have the following content: * R1 001.fastq.gz : contains the three prime UTR cDNA read * R2 001.fastq.gz : contains the first half of the cell barcode * R3 001.fastq.gz : contains the library index for demultiplexing libraries * R4 001.fastq.gz : contains the second half of the barcode and the UMI. All subsequent processing steps from FASTQ files to count matrixes were performed using the custom pipeline for inDrops data analysis github.com/indrops. Single cell transcriptomes were barcoded using inDrops Klein et al Cell 2015. Standard transcriptome RNA seq libraries were processed as reported in Zilionis et al. Nature Protocol 2016 using inDrops v3 protocol. The transcriptome libraries were sequenced on reads Illumina NextSeq 500. Libraries used standard Illumina sequencing primers and 61 cycles for Read1 14 cycles for Read2 8 cycles each for IndexRead1 and IndexRead2. Raw fastq files was processed using inDrops.py pipeline github.com/indrops/indrops. Sequenced reads were mapped to a zebrafish reference transcriptome built from the zebrafish GRCz10 genome assembly Assembly Accession: GCF 000002035.5 using bowtie version 1.1.143. To obtain the final counts matrix used for data analysis total count filters were applied as described in Kukreja et. al. 2024 method section "Single cell RNA seq Data preprocessing" Assembly: GRCz10 genome assembly Assembly Accession: GCF 000002035.5 Supplementary files format and content: Raw counts tsv.gz: cell ba… whole embryo Zebrafish embryos were arrested for cell cycle using two complementary approaches. S phase cell cycle arrest was induced using a cocktail of drugs – hydroxyurea Sigma Aldrich H8627 5G at 20 mM and aphidicolin Sigma Aldrich A0781 5MG at 150 µM concentration in 1% dimethyl sulfoxide DMSO in egg water. G2/M phase arrest was induced by crossing heterozygous emi1 wt/mut zebrafish to generate homozygous emi1 mut/mut embryos referred as hi2648 in the manuscript. Homozygous mutants with cell cycle arrest were screened at 24 hpf as they have very clear phenotype by this time – these embryos are smaller and have bent tails and the heterozygous mutants and wildtype are indistinguishable. Zebrafish embryos were dechorionated using 1mg/mL Pronase Sigma P5147 1G for 5 7 minutes followed by washing in egg water. Embryos were dissociated as previously described in Wagner et. al. Science 2018. Briefly embryos were dissociated in 0.5mL LoBind microcentrifuge tubes that had been precoated with 10% w/v BSA for about 15 minutes at room temperature. They were homogenized in 1XDPBS/1% w/v BSA for 6 hpf to 10 hpf embryos early time points and in 500 µL FACSmax cell dissociation solution Genlantis T200100 for 14 hpf to 24 hpf embryos late time points. Cells were then filtered through a 40µm cell strainer mesh Fisher 352340 and centrifuged in a swinging bucket rotor at 200g for 1 minute early time points or 300g for 5 minutes late time points. Cells were then washed in 1XDPBS/1%BSA using the same centrifuge settings. They were then resuspended in 1XDPBS/0.5%BSA/18% optiprep density medium Sigma D1556 250ML. Cell concentration was manually quantified using hemocytometer and adjusted to a final concentration of 100 000 cells/mL before encapsulation. Library construction as detailed in Zilionis R. et al. 2016 Nature Protocols. Single cell barcoding and sequencing was done using droplet microfluidics also described in the same paper. AB wild type strains were used for all HUA perturbation experiments. Zebrafish mutant line hi2648 a mutant for the gene emi1 was kindly gifted by Dr. Jennifer Rhodes. Embryos were developed within the ambient temperature of Zebrafish development. Time of fertilization at 28.5 C was used to stage each clutch and this was confirmed using morphological features of the embryos. All zebrafish were housed in a facility overseen by the Harvard Medical Area Standing Committee on Animals our IACUC which performs regular inspections and under which we have an approved protocol for all animal procedures. tissue:whole embryo|treatment:1percent DMSO control and cell cycle arrest at 6 hpf GSM8327217 GSM8327217: Perturbation experiment 2 24 hpf biological replicate 2 technical replicate 1 to 6; Danio rerio; OTHER GSM8327217 r1 GSM8327217 1 Zebrafish embryos were dechorionated using 1mg/mL Pronase Sigma P5147 1G for 5 7 minutes followed by washing in egg water. Embryos were dissociated as previously described in Wagner et. al. Science 2018. Briefly embryos were dissociated in 0.5mL LoBind microcentrifuge tubes that had been precoated with 10% w/v BSA for about 15 minutes at room temperature. They were homogenized in 1XDPBS/1% w/v BSA for 6 hpf to 10 hpf embryos early time points and in 500 µL FACSmax cell dissociation solution Genlantis T200100 for 14 hpf to 24 hpf embryos late time points. Cells were then filtered through a 40µm cell strainer mesh Fisher 352340 and centrifuged in a swinging bucket rotor at 200g for 1 minute early time points or 300g for 5 minutes late time points. Cells were then washed in 1XDPBS/1%BSA using the same centrifuge settings. They were then resuspended in 1XDPBS/0.5%BSA/18% optiprep density medium Sigma D1556 250ML. Cell concentration was manually quantified using hemocytometer and adjusted to a final concentration of 100 000 cells/mL before encapsulation. Library construction as detailed in Zilionis R. et al. 2016 Nature Protocols. Single cell barcoding and sequencing was done using droplet microfluidics also described in the same paper.   OTHER TRANSCRIPTOMIC other PAIRED ILLUMINA NextSeq 500   SRP513754     pert_exp2_brep2_trep1_to_6_S0_L002_R1_001.fastq.gz pert_exp2_brep2_trep1_to_6_S0_L002_R2_001.fastq.gz pert_exp2_brep2_trep1_to_6_S0_L002_R3_001.fastq.gz pert_exp2_brep2_trep1_to_6_S0_L002_R4_001.fastq.gz fastq fastq fastq fastq 9113682123.0 100150353.0 GSM8327217 r2 0:61 1:8 2:8 3:14 A:1686097187;C:1259089439;G:1218258982;T:1945625439;N:100486 61 8 8 14 1686097187 1259089439 1218258982 1945625439 100486 SRX24912668 SRS21618601 SRA1898111 Harvard University Harvard University                       B   usable mapping rate illumina nextseq unknown other trueseq sc single_cell_droplet indrops   United States 2024-06-13 Multi-stage Embryo Whole Organism All anatomical structures
32739 32739 SRR29398878 SRX24912668 SRS21618601 SRP513754 PRJNA1123686 Cell state transitions are decoupled from cell division during early embryo development [I] GSE269784 Other As tissues develop cells divide and differentiate concurrently. Conflicting evidence shows that cell division is either dispensable or required for formation of cell types. To determine the role of cell division in differentiation we arrested the cell cycle in zebrafish embryos using two independent approaches and profiled them at single cell resolution. We show that cell division is dispensable for differentiation of all embryonic tissues during initial cell type differentiation from early gastrulation to the end of segmentation. However in the absence of cell division differentiation slows down in some cell types and cells exhibit global stress responses. While differentiation is robust to blocking cell division the proportions of cells across cell states are not but show evidence of partial compensation. This work clarifies our understanding of the role of cell division in development and showcases the utility of combining embryo wide perturbations with single cell RNA sequencing to uncover the role of common biological processes across multiple tissues. Overall design: We arrested the cell cycle in zebrafish embryos at 6 hpf using two independent approaches: a chemical approach hydroxyurea and aphidicolin HUA and a genetic approach involving a loss of function mutation in the gene emi1 allele hi2648. We tracked differentiation dynamics using single cell RNA sequencing scRNA seq on embryos spanning 6–24 hpf for HUA treated and control embryos and at 24 hpf for emi1 mutant embryos. Overall we have collected multiple replicates for control embryos ABs at 6 8 10 14 18 21 hpf and 24 hpf for HUA treated at 8 10 14 hpf and 24 hpf and for emi1 mutants at 24 hpf. Details about the samples can be found in the supplementary file "sample information.txt"   pubmed:37546736   Perturbation experiment 2 24 hpf biological replicate 2 technical replicate 1 to 6 GSM8327217   source name:whole embryo|tissue:whole embryo|treatment:1percent DMSO control and cell cycle arrest at 6 hpf|geo loc name:missing|collection date:missing Perturbation experiment 2 24 hpf biological replicate 2 technical replicate 1 to 6 Multiple samples are pooled for each sequencing run. Raw FASTQ were prepared from Illumina bcl files in a format compatible with the inDrops.py pipeline. Each nextseq run results in 16 FASTQ files 4 lanes x 4 reads per lane. For some pooled libraries sequencing was run twice to get more UMIs per cell and hence we have deposited 16x2 = 32 raw files for those sequencing samples. The illumina library indices of different samples in a run are provided in the meta data. These can be used to demultiplex the raw file into separate libraries. The read files from an inDrops run have the following content: * R1 001.fastq.gz : contains the three prime UTR cDNA read * R2 001.fastq.gz : contains the first half of the cell barcode * R3 001.fastq.gz : contains the library index for demultiplexing libraries * R4 001.fastq.gz : contains the second half of the barcode and the UMI. All subsequent processing steps from FASTQ files to count matrixes were performed using the custom pipeline for inDrops data analysis github.com/indrops. Single cell transcriptomes were barcoded using inDrops Klein et al Cell 2015. Standard transcriptome RNA seq libraries were processed as reported in Zilionis et al. Nature Protocol 2016 using inDrops v3 protocol. The transcriptome libraries were sequenced on reads Illumina NextSeq 500. Libraries used standard Illumina sequencing primers and 61 cycles for Read1 14 cycles for Read2 8 cycles each for IndexRead1 and IndexRead2. Raw fastq files was processed using inDrops.py pipeline github.com/indrops/indrops. Sequenced reads were mapped to a zebrafish reference transcriptome built from the zebrafish GRCz10 genome assembly Assembly Accession: GCF 000002035.5 using bowtie version 1.1.143. To obtain the final counts matrix used for data analysis total count filters were applied as described in Kukreja et. al. 2024 method section "Single cell RNA seq Data preprocessing" Assembly: GRCz10 genome assembly Assembly Accession: GCF 000002035.5 Supplementary files format and content: Raw counts tsv.gz: cell ba… whole embryo Zebrafish embryos were arrested for cell cycle using two complementary approaches. S phase cell cycle arrest was induced using a cocktail of drugs – hydroxyurea Sigma Aldrich H8627 5G at 20 mM and aphidicolin Sigma Aldrich A0781 5MG at 150 µM concentration in 1% dimethyl sulfoxide DMSO in egg water. G2/M phase arrest was induced by crossing heterozygous emi1 wt/mut zebrafish to generate homozygous emi1 mut/mut embryos referred as hi2648 in the manuscript. Homozygous mutants with cell cycle arrest were screened at 24 hpf as they have very clear phenotype by this time – these embryos are smaller and have bent tails and the heterozygous mutants and wildtype are indistinguishable. Zebrafish embryos were dechorionated using 1mg/mL Pronase Sigma P5147 1G for 5 7 minutes followed by washing in egg water. Embryos were dissociated as previously described in Wagner et. al. Science 2018. Briefly embryos were dissociated in 0.5mL LoBind microcentrifuge tubes that had been precoated with 10% w/v BSA for about 15 minutes at room temperature. They were homogenized in 1XDPBS/1% w/v BSA for 6 hpf to 10 hpf embryos early time points and in 500 µL FACSmax cell dissociation solution Genlantis T200100 for 14 hpf to 24 hpf embryos late time points. Cells were then filtered through a 40µm cell strainer mesh Fisher 352340 and centrifuged in a swinging bucket rotor at 200g for 1 minute early time points or 300g for 5 minutes late time points. Cells were then washed in 1XDPBS/1%BSA using the same centrifuge settings. They were then resuspended in 1XDPBS/0.5%BSA/18% optiprep density medium Sigma D1556 250ML. Cell concentration was manually quantified using hemocytometer and adjusted to a final concentration of 100 000 cells/mL before encapsulation. Library construction as detailed in Zilionis R. et al. 2016 Nature Protocols. Single cell barcoding and sequencing was done using droplet microfluidics also described in the same paper. AB wild type strains were used for all HUA perturbation experiments. Zebrafish mutant line hi2648 a mutant for the gene emi1 was kindly gifted by Dr. Jennifer Rhodes. Embryos were developed within the ambient temperature of Zebrafish development. Time of fertilization at 28.5 C was used to stage each clutch and this was confirmed using morphological features of the embryos. All zebrafish were housed in a facility overseen by the Harvard Medical Area Standing Committee on Animals our IACUC which performs regular inspections and under which we have an approved protocol for all animal procedures. tissue:whole embryo|treatment:1percent DMSO control and cell cycle arrest at 6 hpf GSM8327217 GSM8327217: Perturbation experiment 2 24 hpf biological replicate 2 technical replicate 1 to 6; Danio rerio; OTHER GSM8327217 r1 GSM8327217 1 Zebrafish embryos were dechorionated using 1mg/mL Pronase Sigma P5147 1G for 5 7 minutes followed by washing in egg water. Embryos were dissociated as previously described in Wagner et. al. Science 2018. Briefly embryos were dissociated in 0.5mL LoBind microcentrifuge tubes that had been precoated with 10% w/v BSA for about 15 minutes at room temperature. They were homogenized in 1XDPBS/1% w/v BSA for 6 hpf to 10 hpf embryos early time points and in 500 µL FACSmax cell dissociation solution Genlantis T200100 for 14 hpf to 24 hpf embryos late time points. Cells were then filtered through a 40µm cell strainer mesh Fisher 352340 and centrifuged in a swinging bucket rotor at 200g for 1 minute early time points or 300g for 5 minutes late time points. Cells were then washed in 1XDPBS/1%BSA using the same centrifuge settings. They were then resuspended in 1XDPBS/0.5%BSA/18% optiprep density medium Sigma D1556 250ML. Cell concentration was manually quantified using hemocytometer and adjusted to a final concentration of 100 000 cells/mL before encapsulation. Library construction as detailed in Zilionis R. et al. 2016 Nature Protocols. Single cell barcoding and sequencing was done using droplet microfluidics also described in the same paper.   OTHER TRANSCRIPTOMIC other PAIRED ILLUMINA NextSeq 500   SRP513754     pert_exp2_brep2_trep1_to_6_S0_L003_R1_001.fastq.gz pert_exp2_brep2_trep1_to_6_S0_L003_R2_001.fastq.gz pert_exp2_brep2_trep1_to_6_S0_L003_R3_001.fastq.gz pert_exp2_brep2_trep1_to_6_S0_L003_R4_001.fastq.gz fastq fastq fastq fastq 9286087720.0 102044920.0 GSM8327217 r3 0:61 1:8 2:8 3:14 A:1716420906;C:1285666517;G:1243365905;T:1979187027;N:99765 61 8 8 14 1716420906 1285666517 1243365905 1979187027 99765 SRX24912668 SRS21618601 SRA1898111 Harvard University Harvard University                       B   usable mapping rate illumina nextseq unknown other trueseq sc single_cell_droplet indrops   United States 2024-06-13 Multi-stage Embryo Whole Organism All anatomical structures
32740 32740 SRR29398879 SRX24912668 SRS21618601 SRP513754 PRJNA1123686 Cell state transitions are decoupled from cell division during early embryo development [I] GSE269784 Other As tissues develop cells divide and differentiate concurrently. Conflicting evidence shows that cell division is either dispensable or required for formation of cell types. To determine the role of cell division in differentiation we arrested the cell cycle in zebrafish embryos using two independent approaches and profiled them at single cell resolution. We show that cell division is dispensable for differentiation of all embryonic tissues during initial cell type differentiation from early gastrulation to the end of segmentation. However in the absence of cell division differentiation slows down in some cell types and cells exhibit global stress responses. While differentiation is robust to blocking cell division the proportions of cells across cell states are not but show evidence of partial compensation. This work clarifies our understanding of the role of cell division in development and showcases the utility of combining embryo wide perturbations with single cell RNA sequencing to uncover the role of common biological processes across multiple tissues. Overall design: We arrested the cell cycle in zebrafish embryos at 6 hpf using two independent approaches: a chemical approach hydroxyurea and aphidicolin HUA and a genetic approach involving a loss of function mutation in the gene emi1 allele hi2648. We tracked differentiation dynamics using single cell RNA sequencing scRNA seq on embryos spanning 6–24 hpf for HUA treated and control embryos and at 24 hpf for emi1 mutant embryos. Overall we have collected multiple replicates for control embryos ABs at 6 8 10 14 18 21 hpf and 24 hpf for HUA treated at 8 10 14 hpf and 24 hpf and for emi1 mutants at 24 hpf. Details about the samples can be found in the supplementary file "sample information.txt"   pubmed:37546736   Perturbation experiment 2 24 hpf biological replicate 2 technical replicate 1 to 6 GSM8327217   source name:whole embryo|tissue:whole embryo|treatment:1percent DMSO control and cell cycle arrest at 6 hpf|geo loc name:missing|collection date:missing Perturbation experiment 2 24 hpf biological replicate 2 technical replicate 1 to 6 Multiple samples are pooled for each sequencing run. Raw FASTQ were prepared from Illumina bcl files in a format compatible with the inDrops.py pipeline. Each nextseq run results in 16 FASTQ files 4 lanes x 4 reads per lane. For some pooled libraries sequencing was run twice to get more UMIs per cell and hence we have deposited 16x2 = 32 raw files for those sequencing samples. The illumina library indices of different samples in a run are provided in the meta data. These can be used to demultiplex the raw file into separate libraries. The read files from an inDrops run have the following content: * R1 001.fastq.gz : contains the three prime UTR cDNA read * R2 001.fastq.gz : contains the first half of the cell barcode * R3 001.fastq.gz : contains the library index for demultiplexing libraries * R4 001.fastq.gz : contains the second half of the barcode and the UMI. All subsequent processing steps from FASTQ files to count matrixes were performed using the custom pipeline for inDrops data analysis github.com/indrops. Single cell transcriptomes were barcoded using inDrops Klein et al Cell 2015. Standard transcriptome RNA seq libraries were processed as reported in Zilionis et al. Nature Protocol 2016 using inDrops v3 protocol. The transcriptome libraries were sequenced on reads Illumina NextSeq 500. Libraries used standard Illumina sequencing primers and 61 cycles for Read1 14 cycles for Read2 8 cycles each for IndexRead1 and IndexRead2. Raw fastq files was processed using inDrops.py pipeline github.com/indrops/indrops. Sequenced reads were mapped to a zebrafish reference transcriptome built from the zebrafish GRCz10 genome assembly Assembly Accession: GCF 000002035.5 using bowtie version 1.1.143. To obtain the final counts matrix used for data analysis total count filters were applied as described in Kukreja et. al. 2024 method section "Single cell RNA seq Data preprocessing" Assembly: GRCz10 genome assembly Assembly Accession: GCF 000002035.5 Supplementary files format and content: Raw counts tsv.gz: cell ba… whole embryo Zebrafish embryos were arrested for cell cycle using two complementary approaches. S phase cell cycle arrest was induced using a cocktail of drugs – hydroxyurea Sigma Aldrich H8627 5G at 20 mM and aphidicolin Sigma Aldrich A0781 5MG at 150 µM concentration in 1% dimethyl sulfoxide DMSO in egg water. G2/M phase arrest was induced by crossing heterozygous emi1 wt/mut zebrafish to generate homozygous emi1 mut/mut embryos referred as hi2648 in the manuscript. Homozygous mutants with cell cycle arrest were screened at 24 hpf as they have very clear phenotype by this time – these embryos are smaller and have bent tails and the heterozygous mutants and wildtype are indistinguishable. Zebrafish embryos were dechorionated using 1mg/mL Pronase Sigma P5147 1G for 5 7 minutes followed by washing in egg water. Embryos were dissociated as previously described in Wagner et. al. Science 2018. Briefly embryos were dissociated in 0.5mL LoBind microcentrifuge tubes that had been precoated with 10% w/v BSA for about 15 minutes at room temperature. They were homogenized in 1XDPBS/1% w/v BSA for 6 hpf to 10 hpf embryos early time points and in 500 µL FACSmax cell dissociation solution Genlantis T200100 for 14 hpf to 24 hpf embryos late time points. Cells were then filtered through a 40µm cell strainer mesh Fisher 352340 and centrifuged in a swinging bucket rotor at 200g for 1 minute early time points or 300g for 5 minutes late time points. Cells were then washed in 1XDPBS/1%BSA using the same centrifuge settings. They were then resuspended in 1XDPBS/0.5%BSA/18% optiprep density medium Sigma D1556 250ML. Cell concentration was manually quantified using hemocytometer and adjusted to a final concentration of 100 000 cells/mL before encapsulation. Library construction as detailed in Zilionis R. et al. 2016 Nature Protocols. Single cell barcoding and sequencing was done using droplet microfluidics also described in the same paper. AB wild type strains were used for all HUA perturbation experiments. Zebrafish mutant line hi2648 a mutant for the gene emi1 was kindly gifted by Dr. Jennifer Rhodes. Embryos were developed within the ambient temperature of Zebrafish development. Time of fertilization at 28.5 C was used to stage each clutch and this was confirmed using morphological features of the embryos. All zebrafish were housed in a facility overseen by the Harvard Medical Area Standing Committee on Animals our IACUC which performs regular inspections and under which we have an approved protocol for all animal procedures. tissue:whole embryo|treatment:1percent DMSO control and cell cycle arrest at 6 hpf GSM8327217 GSM8327217: Perturbation experiment 2 24 hpf biological replicate 2 technical replicate 1 to 6; Danio rerio; OTHER GSM8327217 r1 GSM8327217 1 Zebrafish embryos were dechorionated using 1mg/mL Pronase Sigma P5147 1G for 5 7 minutes followed by washing in egg water. Embryos were dissociated as previously described in Wagner et. al. Science 2018. Briefly embryos were dissociated in 0.5mL LoBind microcentrifuge tubes that had been precoated with 10% w/v BSA for about 15 minutes at room temperature. They were homogenized in 1XDPBS/1% w/v BSA for 6 hpf to 10 hpf embryos early time points and in 500 µL FACSmax cell dissociation solution Genlantis T200100 for 14 hpf to 24 hpf embryos late time points. Cells were then filtered through a 40µm cell strainer mesh Fisher 352340 and centrifuged in a swinging bucket rotor at 200g for 1 minute early time points or 300g for 5 minutes late time points. Cells were then washed in 1XDPBS/1%BSA using the same centrifuge settings. They were then resuspended in 1XDPBS/0.5%BSA/18% optiprep density medium Sigma D1556 250ML. Cell concentration was manually quantified using hemocytometer and adjusted to a final concentration of 100 000 cells/mL before encapsulation. Library construction as detailed in Zilionis R. et al. 2016 Nature Protocols. Single cell barcoding and sequencing was done using droplet microfluidics also described in the same paper.   OTHER TRANSCRIPTOMIC other PAIRED ILLUMINA NextSeq 500   SRP513754     pert_exp2_brep2_trep1_to_6_S0_L004_R1_001.fastq.gz pert_exp2_brep2_trep1_to_6_S0_L004_R2_001.fastq.gz pert_exp2_brep2_trep1_to_6_S0_L004_R3_001.fastq.gz pert_exp2_brep2_trep1_to_6_S0_L004_R4_001.fastq.gz fastq fastq fastq fastq 9274368740.0 101916140.0 GSM8327217 r4 0:61 1:8 2:8 3:14 A:1714501829;C:1283534271;G:1240916416;T:1977833740;N:98284 61 8 8 14 1714501829 1283534271 1240916416 1977833740 98284 SRX24912668 SRS21618601 SRA1898111 Harvard University Harvard University                       B   usable mapping rate illumina nextseq unknown other trueseq sc single_cell_droplet indrops   United States 2024-06-13 Multi-stage Embryo Whole Organism All anatomical structures
32741 32741 SRR29398880 SRX24912667 SRS21618602 SRP513754 PRJNA1123686 Cell state transitions are decoupled from cell division during early embryo development [I] GSE269784 Other As tissues develop cells divide and differentiate concurrently. Conflicting evidence shows that cell division is either dispensable or required for formation of cell types. To determine the role of cell division in differentiation we arrested the cell cycle in zebrafish embryos using two independent approaches and profiled them at single cell resolution. We show that cell division is dispensable for differentiation of all embryonic tissues during initial cell type differentiation from early gastrulation to the end of segmentation. However in the absence of cell division differentiation slows down in some cell types and cells exhibit global stress responses. While differentiation is robust to blocking cell division the proportions of cells across cell states are not but show evidence of partial compensation. This work clarifies our understanding of the role of cell division in development and showcases the utility of combining embryo wide perturbations with single cell RNA sequencing to uncover the role of common biological processes across multiple tissues. Overall design: We arrested the cell cycle in zebrafish embryos at 6 hpf using two independent approaches: a chemical approach hydroxyurea and aphidicolin HUA and a genetic approach involving a loss of function mutation in the gene emi1 allele hi2648. We tracked differentiation dynamics using single cell RNA sequencing scRNA seq on embryos spanning 6–24 hpf for HUA treated and control embryos and at 24 hpf for emi1 mutant embryos. Overall we have collected multiple replicates for control embryos ABs at 6 8 10 14 18 21 hpf and 24 hpf for HUA treated at 8 10 14 hpf and 24 hpf and for emi1 mutants at 24 hpf. Details about the samples can be found in the supplementary file "sample information.txt"   pubmed:37546736   Perturbation experiment 2 24 hpf biological replicate 1 technical replicate 1 to 4 GSM8327216   source name:whole embryo|tissue:whole embryo|treatment:1percent DMSO control and cell cycle arrest at 6 hpf|geo loc name:missing|collection date:missing Perturbation experiment 2 24 hpf biological replicate 1 technical replicate 1 to 4 Multiple samples are pooled for each sequencing run. Raw FASTQ were prepared from Illumina bcl files in a format compatible with the inDrops.py pipeline. Each nextseq run results in 16 FASTQ files 4 lanes x 4 reads per lane. For some pooled libraries sequencing was run twice to get more UMIs per cell and hence we have deposited 16x2 = 32 raw files for those sequencing samples. The illumina library indices of different samples in a run are provided in the meta data. These can be used to demultiplex the raw file into separate libraries. The read files from an inDrops run have the following content: * R1 001.fastq.gz : contains the three prime UTR cDNA read * R2 001.fastq.gz : contains the first half of the cell barcode * R3 001.fastq.gz : contains the library index for demultiplexing libraries * R4 001.fastq.gz : contains the second half of the barcode and the UMI. All subsequent processing steps from FASTQ files to count matrixes were performed using the custom pipeline for inDrops data analysis github.com/indrops. Single cell transcriptomes were barcoded using inDrops Klein et al Cell 2015. Standard transcriptome RNA seq libraries were processed as reported in Zilionis et al. Nature Protocol 2016 using inDrops v3 protocol. The transcriptome libraries were sequenced on reads Illumina NextSeq 500. Libraries used standard Illumina sequencing primers and 61 cycles for Read1 14 cycles for Read2 8 cycles each for IndexRead1 and IndexRead2. Raw fastq files was processed using inDrops.py pipeline github.com/indrops/indrops. Sequenced reads were mapped to a zebrafish reference transcriptome built from the zebrafish GRCz10 genome assembly Assembly Accession: GCF 000002035.5 using bowtie version 1.1.143. To obtain the final counts matrix used for data analysis total count filters were applied as described in Kukreja et. al. 2024 method section "Single cell RNA seq Data preprocessing" Assembly: GRCz10 genome assembly Assembly Accession: GCF 000002035.5 Supplementary files format and content: Raw counts tsv.gz: cell ba… whole embryo Zebrafish embryos were arrested for cell cycle using two complementary approaches. S phase cell cycle arrest was induced using a cocktail of drugs – hydroxyurea Sigma Aldrich H8627 5G at 20 mM and aphidicolin Sigma Aldrich A0781 5MG at 150 µM concentration in 1% dimethyl sulfoxide DMSO in egg water. G2/M phase arrest was induced by crossing heterozygous emi1 wt/mut zebrafish to generate homozygous emi1 mut/mut embryos referred as hi2648 in the manuscript. Homozygous mutants with cell cycle arrest were screened at 24 hpf as they have very clear phenotype by this time – these embryos are smaller and have bent tails and the heterozygous mutants and wildtype are indistinguishable. Zebrafish embryos were dechorionated using 1mg/mL Pronase Sigma P5147 1G for 5 7 minutes followed by washing in egg water. Embryos were dissociated as previously described in Wagner et. al. Science 2018. Briefly embryos were dissociated in 0.5mL LoBind microcentrifuge tubes that had been precoated with 10% w/v BSA for about 15 minutes at room temperature. They were homogenized in 1XDPBS/1% w/v BSA for 6 hpf to 10 hpf embryos early time points and in 500 µL FACSmax cell dissociation solution Genlantis T200100 for 14 hpf to 24 hpf embryos late time points. Cells were then filtered through a 40µm cell strainer mesh Fisher 352340 and centrifuged in a swinging bucket rotor at 200g for 1 minute early time points or 300g for 5 minutes late time points. Cells were then washed in 1XDPBS/1%BSA using the same centrifuge settings. They were then resuspended in 1XDPBS/0.5%BSA/18% optiprep density medium Sigma D1556 250ML. Cell concentration was manually quantified using hemocytometer and adjusted to a final concentration of 100 000 cells/mL before encapsulation. Library construction as detailed in Zilionis R. et al. 2016 Nature Protocols. Single cell barcoding and sequencing was done using droplet microfluidics also described in the same paper. AB wild type strains were used for all HUA perturbation experiments. Zebrafish mutant line hi2648 a mutant for the gene emi1 was kindly gifted by Dr. Jennifer Rhodes. Embryos were developed within the ambient temperature of Zebrafish development. Time of fertilization at 28.5 C was used to stage each clutch and this was confirmed using morphological features of the embryos. All zebrafish were housed in a facility overseen by the Harvard Medical Area Standing Committee on Animals our IACUC which performs regular inspections and under which we have an approved protocol for all animal procedures. tissue:whole embryo|treatment:1percent DMSO control and cell cycle arrest at 6 hpf GSM8327216 GSM8327216: Perturbation experiment 2 24 hpf biological replicate 1 technical replicate 1 to 4; Danio rerio; OTHER GSM8327216 r1 GSM8327216 1 Zebrafish embryos were dechorionated using 1mg/mL Pronase Sigma P5147 1G for 5 7 minutes followed by washing in egg water. Embryos were dissociated as previously described in Wagner et. al. Science 2018. Briefly embryos were dissociated in 0.5mL LoBind microcentrifuge tubes that had been precoated with 10% w/v BSA for about 15 minutes at room temperature. They were homogenized in 1XDPBS/1% w/v BSA for 6 hpf to 10 hpf embryos early time points and in 500 µL FACSmax cell dissociation solution Genlantis T200100 for 14 hpf to 24 hpf embryos late time points. Cells were then filtered through a 40µm cell strainer mesh Fisher 352340 and centrifuged in a swinging bucket rotor at 200g for 1 minute early time points or 300g for 5 minutes late time points. Cells were then washed in 1XDPBS/1%BSA using the same centrifuge settings. They were then resuspended in 1XDPBS/0.5%BSA/18% optiprep density medium Sigma D1556 250ML. Cell concentration was manually quantified using hemocytometer and adjusted to a final concentration of 100 000 cells/mL before encapsulation. Library construction as detailed in Zilionis R. et al. 2016 Nature Protocols. Single cell barcoding and sequencing was done using droplet microfluidics also described in the same paper.   OTHER TRANSCRIPTOMIC other PAIRED ILLUMINA NextSeq 500   SRP513754     pert_exp2_brep1_trep1_2_3_4_S0_L001_R1_001.fastq.gz pert_exp2_brep1_trep1_2_3_4_S0_L001_R2_001.fastq.gz pert_exp2_brep1_trep1_2_3_4_S0_L001_R3_001.fastq.gz pert_exp2_brep1_trep1_2_3_4_S0_L001_R4_001.fastq.gz fastq fastq fastq fastq 10478915720.0 115152920.0 GSM8327216 r1 0:61 1:8 2:8 3:14 A:1736895270;C:1397254088;G:1357881943;T:2531770836;N:525983 61 8 8 14 1736895270 1397254088 1357881943 2531770836 525983 SRX24912667 SRS21618602 SRA1898111 Harvard University Harvard University                       B   usable mapping rate illumina nextseq unknown other trueseq sc single_cell_droplet indrops   United States 2024-06-13 Multi-stage Embryo Whole Organism All anatomical structures
32742 32742 SRR29398881 SRX24912667 SRS21618602 SRP513754 PRJNA1123686 Cell state transitions are decoupled from cell division during early embryo development [I] GSE269784 Other As tissues develop cells divide and differentiate concurrently. Conflicting evidence shows that cell division is either dispensable or required for formation of cell types. To determine the role of cell division in differentiation we arrested the cell cycle in zebrafish embryos using two independent approaches and profiled them at single cell resolution. We show that cell division is dispensable for differentiation of all embryonic tissues during initial cell type differentiation from early gastrulation to the end of segmentation. However in the absence of cell division differentiation slows down in some cell types and cells exhibit global stress responses. While differentiation is robust to blocking cell division the proportions of cells across cell states are not but show evidence of partial compensation. This work clarifies our understanding of the role of cell division in development and showcases the utility of combining embryo wide perturbations with single cell RNA sequencing to uncover the role of common biological processes across multiple tissues. Overall design: We arrested the cell cycle in zebrafish embryos at 6 hpf using two independent approaches: a chemical approach hydroxyurea and aphidicolin HUA and a genetic approach involving a loss of function mutation in the gene emi1 allele hi2648. We tracked differentiation dynamics using single cell RNA sequencing scRNA seq on embryos spanning 6–24 hpf for HUA treated and control embryos and at 24 hpf for emi1 mutant embryos. Overall we have collected multiple replicates for control embryos ABs at 6 8 10 14 18 21 hpf and 24 hpf for HUA treated at 8 10 14 hpf and 24 hpf and for emi1 mutants at 24 hpf. Details about the samples can be found in the supplementary file "sample information.txt"   pubmed:37546736   Perturbation experiment 2 24 hpf biological replicate 1 technical replicate 1 to 4 GSM8327216   source name:whole embryo|tissue:whole embryo|treatment:1percent DMSO control and cell cycle arrest at 6 hpf|geo loc name:missing|collection date:missing Perturbation experiment 2 24 hpf biological replicate 1 technical replicate 1 to 4 Multiple samples are pooled for each sequencing run. Raw FASTQ were prepared from Illumina bcl files in a format compatible with the inDrops.py pipeline. Each nextseq run results in 16 FASTQ files 4 lanes x 4 reads per lane. For some pooled libraries sequencing was run twice to get more UMIs per cell and hence we have deposited 16x2 = 32 raw files for those sequencing samples. The illumina library indices of different samples in a run are provided in the meta data. These can be used to demultiplex the raw file into separate libraries. The read files from an inDrops run have the following content: * R1 001.fastq.gz : contains the three prime UTR cDNA read * R2 001.fastq.gz : contains the first half of the cell barcode * R3 001.fastq.gz : contains the library index for demultiplexing libraries * R4 001.fastq.gz : contains the second half of the barcode and the UMI. All subsequent processing steps from FASTQ files to count matrixes were performed using the custom pipeline for inDrops data analysis github.com/indrops. Single cell transcriptomes were barcoded using inDrops Klein et al Cell 2015. Standard transcriptome RNA seq libraries were processed as reported in Zilionis et al. Nature Protocol 2016 using inDrops v3 protocol. The transcriptome libraries were sequenced on reads Illumina NextSeq 500. Libraries used standard Illumina sequencing primers and 61 cycles for Read1 14 cycles for Read2 8 cycles each for IndexRead1 and IndexRead2. Raw fastq files was processed using inDrops.py pipeline github.com/indrops/indrops. Sequenced reads were mapped to a zebrafish reference transcriptome built from the zebrafish GRCz10 genome assembly Assembly Accession: GCF 000002035.5 using bowtie version 1.1.143. To obtain the final counts matrix used for data analysis total count filters were applied as described in Kukreja et. al. 2024 method section "Single cell RNA seq Data preprocessing" Assembly: GRCz10 genome assembly Assembly Accession: GCF 000002035.5 Supplementary files format and content: Raw counts tsv.gz: cell ba… whole embryo Zebrafish embryos were arrested for cell cycle using two complementary approaches. S phase cell cycle arrest was induced using a cocktail of drugs – hydroxyurea Sigma Aldrich H8627 5G at 20 mM and aphidicolin Sigma Aldrich A0781 5MG at 150 µM concentration in 1% dimethyl sulfoxide DMSO in egg water. G2/M phase arrest was induced by crossing heterozygous emi1 wt/mut zebrafish to generate homozygous emi1 mut/mut embryos referred as hi2648 in the manuscript. Homozygous mutants with cell cycle arrest were screened at 24 hpf as they have very clear phenotype by this time – these embryos are smaller and have bent tails and the heterozygous mutants and wildtype are indistinguishable. Zebrafish embryos were dechorionated using 1mg/mL Pronase Sigma P5147 1G for 5 7 minutes followed by washing in egg water. Embryos were dissociated as previously described in Wagner et. al. Science 2018. Briefly embryos were dissociated in 0.5mL LoBind microcentrifuge tubes that had been precoated with 10% w/v BSA for about 15 minutes at room temperature. They were homogenized in 1XDPBS/1% w/v BSA for 6 hpf to 10 hpf embryos early time points and in 500 µL FACSmax cell dissociation solution Genlantis T200100 for 14 hpf to 24 hpf embryos late time points. Cells were then filtered through a 40µm cell strainer mesh Fisher 352340 and centrifuged in a swinging bucket rotor at 200g for 1 minute early time points or 300g for 5 minutes late time points. Cells were then washed in 1XDPBS/1%BSA using the same centrifuge settings. They were then resuspended in 1XDPBS/0.5%BSA/18% optiprep density medium Sigma D1556 250ML. Cell concentration was manually quantified using hemocytometer and adjusted to a final concentration of 100 000 cells/mL before encapsulation. Library construction as detailed in Zilionis R. et al. 2016 Nature Protocols. Single cell barcoding and sequencing was done using droplet microfluidics also described in the same paper. AB wild type strains were used for all HUA perturbation experiments. Zebrafish mutant line hi2648 a mutant for the gene emi1 was kindly gifted by Dr. Jennifer Rhodes. Embryos were developed within the ambient temperature of Zebrafish development. Time of fertilization at 28.5 C was used to stage each clutch and this was confirmed using morphological features of the embryos. All zebrafish were housed in a facility overseen by the Harvard Medical Area Standing Committee on Animals our IACUC which performs regular inspections and under which we have an approved protocol for all animal procedures. tissue:whole embryo|treatment:1percent DMSO control and cell cycle arrest at 6 hpf GSM8327216 GSM8327216: Perturbation experiment 2 24 hpf biological replicate 1 technical replicate 1 to 4; Danio rerio; OTHER GSM8327216 r1 GSM8327216 1 Zebrafish embryos were dechorionated using 1mg/mL Pronase Sigma P5147 1G for 5 7 minutes followed by washing in egg water. Embryos were dissociated as previously described in Wagner et. al. Science 2018. Briefly embryos were dissociated in 0.5mL LoBind microcentrifuge tubes that had been precoated with 10% w/v BSA for about 15 minutes at room temperature. They were homogenized in 1XDPBS/1% w/v BSA for 6 hpf to 10 hpf embryos early time points and in 500 µL FACSmax cell dissociation solution Genlantis T200100 for 14 hpf to 24 hpf embryos late time points. Cells were then filtered through a 40µm cell strainer mesh Fisher 352340 and centrifuged in a swinging bucket rotor at 200g for 1 minute early time points or 300g for 5 minutes late time points. Cells were then washed in 1XDPBS/1%BSA using the same centrifuge settings. They were then resuspended in 1XDPBS/0.5%BSA/18% optiprep density medium Sigma D1556 250ML. Cell concentration was manually quantified using hemocytometer and adjusted to a final concentration of 100 000 cells/mL before encapsulation. Library construction as detailed in Zilionis R. et al. 2016 Nature Protocols. Single cell barcoding and sequencing was done using droplet microfluidics also described in the same paper.   OTHER TRANSCRIPTOMIC other PAIRED ILLUMINA NextSeq 500   SRP513754     pert_exp2_brep1_trep1_2_3_4_S0_L002_R1_001.fastq.gz pert_exp2_brep1_trep1_2_3_4_S0_L002_R2_001.fastq.gz pert_exp2_brep1_trep1_2_3_4_S0_L002_R3_001.fastq.gz pert_exp2_brep1_trep1_2_3_4_S0_L002_R4_001.fastq.gz fastq fastq fastq fastq 10976773171.0 120623881.0 GSM8327216 r2 0:61 1:8 2:8 3:14 A:1854295106;C:1459958363;G:1422957853;T:2620345368;N:500051 61 8 8 14 1854295106 1459958363 1422957853 2620345368 500051 SRX24912667 SRS21618602 SRA1898111 Harvard University Harvard University                       B   usable mapping rate illumina nextseq unknown other trueseq sc single_cell_droplet indrops   United States 2024-06-13 Multi-stage Embryo Whole Organism All anatomical structures
32743 32743 SRR29398882 SRX24912667 SRS21618602 SRP513754 PRJNA1123686 Cell state transitions are decoupled from cell division during early embryo development [I] GSE269784 Other As tissues develop cells divide and differentiate concurrently. Conflicting evidence shows that cell division is either dispensable or required for formation of cell types. To determine the role of cell division in differentiation we arrested the cell cycle in zebrafish embryos using two independent approaches and profiled them at single cell resolution. We show that cell division is dispensable for differentiation of all embryonic tissues during initial cell type differentiation from early gastrulation to the end of segmentation. However in the absence of cell division differentiation slows down in some cell types and cells exhibit global stress responses. While differentiation is robust to blocking cell division the proportions of cells across cell states are not but show evidence of partial compensation. This work clarifies our understanding of the role of cell division in development and showcases the utility of combining embryo wide perturbations with single cell RNA sequencing to uncover the role of common biological processes across multiple tissues. Overall design: We arrested the cell cycle in zebrafish embryos at 6 hpf using two independent approaches: a chemical approach hydroxyurea and aphidicolin HUA and a genetic approach involving a loss of function mutation in the gene emi1 allele hi2648. We tracked differentiation dynamics using single cell RNA sequencing scRNA seq on embryos spanning 6–24 hpf for HUA treated and control embryos and at 24 hpf for emi1 mutant embryos. Overall we have collected multiple replicates for control embryos ABs at 6 8 10 14 18 21 hpf and 24 hpf for HUA treated at 8 10 14 hpf and 24 hpf and for emi1 mutants at 24 hpf. Details about the samples can be found in the supplementary file "sample information.txt"   pubmed:37546736   Perturbation experiment 2 24 hpf biological replicate 1 technical replicate 1 to 4 GSM8327216   source name:whole embryo|tissue:whole embryo|treatment:1percent DMSO control and cell cycle arrest at 6 hpf|geo loc name:missing|collection date:missing Perturbation experiment 2 24 hpf biological replicate 1 technical replicate 1 to 4 Multiple samples are pooled for each sequencing run. Raw FASTQ were prepared from Illumina bcl files in a format compatible with the inDrops.py pipeline. Each nextseq run results in 16 FASTQ files 4 lanes x 4 reads per lane. For some pooled libraries sequencing was run twice to get more UMIs per cell and hence we have deposited 16x2 = 32 raw files for those sequencing samples. The illumina library indices of different samples in a run are provided in the meta data. These can be used to demultiplex the raw file into separate libraries. The read files from an inDrops run have the following content: * R1 001.fastq.gz : contains the three prime UTR cDNA read * R2 001.fastq.gz : contains the first half of the cell barcode * R3 001.fastq.gz : contains the library index for demultiplexing libraries * R4 001.fastq.gz : contains the second half of the barcode and the UMI. All subsequent processing steps from FASTQ files to count matrixes were performed using the custom pipeline for inDrops data analysis github.com/indrops. Single cell transcriptomes were barcoded using inDrops Klein et al Cell 2015. Standard transcriptome RNA seq libraries were processed as reported in Zilionis et al. Nature Protocol 2016 using inDrops v3 protocol. The transcriptome libraries were sequenced on reads Illumina NextSeq 500. Libraries used standard Illumina sequencing primers and 61 cycles for Read1 14 cycles for Read2 8 cycles each for IndexRead1 and IndexRead2. Raw fastq files was processed using inDrops.py pipeline github.com/indrops/indrops. Sequenced reads were mapped to a zebrafish reference transcriptome built from the zebrafish GRCz10 genome assembly Assembly Accession: GCF 000002035.5 using bowtie version 1.1.143. To obtain the final counts matrix used for data analysis total count filters were applied as described in Kukreja et. al. 2024 method section "Single cell RNA seq Data preprocessing" Assembly: GRCz10 genome assembly Assembly Accession: GCF 000002035.5 Supplementary files format and content: Raw counts tsv.gz: cell ba… whole embryo Zebrafish embryos were arrested for cell cycle using two complementary approaches. S phase cell cycle arrest was induced using a cocktail of drugs – hydroxyurea Sigma Aldrich H8627 5G at 20 mM and aphidicolin Sigma Aldrich A0781 5MG at 150 µM concentration in 1% dimethyl sulfoxide DMSO in egg water. G2/M phase arrest was induced by crossing heterozygous emi1 wt/mut zebrafish to generate homozygous emi1 mut/mut embryos referred as hi2648 in the manuscript. Homozygous mutants with cell cycle arrest were screened at 24 hpf as they have very clear phenotype by this time – these embryos are smaller and have bent tails and the heterozygous mutants and wildtype are indistinguishable. Zebrafish embryos were dechorionated using 1mg/mL Pronase Sigma P5147 1G for 5 7 minutes followed by washing in egg water. Embryos were dissociated as previously described in Wagner et. al. Science 2018. Briefly embryos were dissociated in 0.5mL LoBind microcentrifuge tubes that had been precoated with 10% w/v BSA for about 15 minutes at room temperature. They were homogenized in 1XDPBS/1% w/v BSA for 6 hpf to 10 hpf embryos early time points and in 500 µL FACSmax cell dissociation solution Genlantis T200100 for 14 hpf to 24 hpf embryos late time points. Cells were then filtered through a 40µm cell strainer mesh Fisher 352340 and centrifuged in a swinging bucket rotor at 200g for 1 minute early time points or 300g for 5 minutes late time points. Cells were then washed in 1XDPBS/1%BSA using the same centrifuge settings. They were then resuspended in 1XDPBS/0.5%BSA/18% optiprep density medium Sigma D1556 250ML. Cell concentration was manually quantified using hemocytometer and adjusted to a final concentration of 100 000 cells/mL before encapsulation. Library construction as detailed in Zilionis R. et al. 2016 Nature Protocols. Single cell barcoding and sequencing was done using droplet microfluidics also described in the same paper. AB wild type strains were used for all HUA perturbation experiments. Zebrafish mutant line hi2648 a mutant for the gene emi1 was kindly gifted by Dr. Jennifer Rhodes. Embryos were developed within the ambient temperature of Zebrafish development. Time of fertilization at 28.5 C was used to stage each clutch and this was confirmed using morphological features of the embryos. All zebrafish were housed in a facility overseen by the Harvard Medical Area Standing Committee on Animals our IACUC which performs regular inspections and under which we have an approved protocol for all animal procedures. tissue:whole embryo|treatment:1percent DMSO control and cell cycle arrest at 6 hpf GSM8327216 GSM8327216: Perturbation experiment 2 24 hpf biological replicate 1 technical replicate 1 to 4; Danio rerio; OTHER GSM8327216 r1 GSM8327216 1 Zebrafish embryos were dechorionated using 1mg/mL Pronase Sigma P5147 1G for 5 7 minutes followed by washing in egg water. Embryos were dissociated as previously described in Wagner et. al. Science 2018. Briefly embryos were dissociated in 0.5mL LoBind microcentrifuge tubes that had been precoated with 10% w/v BSA for about 15 minutes at room temperature. They were homogenized in 1XDPBS/1% w/v BSA for 6 hpf to 10 hpf embryos early time points and in 500 µL FACSmax cell dissociation solution Genlantis T200100 for 14 hpf to 24 hpf embryos late time points. Cells were then filtered through a 40µm cell strainer mesh Fisher 352340 and centrifuged in a swinging bucket rotor at 200g for 1 minute early time points or 300g for 5 minutes late time points. Cells were then washed in 1XDPBS/1%BSA using the same centrifuge settings. They were then resuspended in 1XDPBS/0.5%BSA/18% optiprep density medium Sigma D1556 250ML. Cell concentration was manually quantified using hemocytometer and adjusted to a final concentration of 100 000 cells/mL before encapsulation. Library construction as detailed in Zilionis R. et al. 2016 Nature Protocols. Single cell barcoding and sequencing was done using droplet microfluidics also described in the same paper.   OTHER TRANSCRIPTOMIC other PAIRED ILLUMINA NextSeq 500   SRP513754     pert_exp2_brep1_trep1_2_3_4_S0_L003_R1_001.fastq.gz pert_exp2_brep1_trep1_2_3_4_S0_L003_R2_001.fastq.gz pert_exp2_brep1_trep1_2_3_4_S0_L003_R3_001.fastq.gz pert_exp2_brep1_trep1_2_3_4_S0_L003_R4_001.fastq.gz fastq fastq fastq fastq 10970493898.0 120554878.0 GSM8327216 r3 0:61 1:8 2:8 3:14 A:1804346523;C:1462166175;G:1421809080;T:2665109964;N:415816 61 8 8 14 1804346523 1462166175 1421809080 2665109964 415816 SRX24912667 SRS21618602 SRA1898111 Harvard University Harvard University                       B   usable mapping rate illumina nextseq unknown other trueseq sc single_cell_droplet indrops   United States 2024-06-13 Multi-stage Embryo Whole Organism All anatomical structures
32744 32744 SRR29398883 SRX24912667 SRS21618602 SRP513754 PRJNA1123686 Cell state transitions are decoupled from cell division during early embryo development [I] GSE269784 Other As tissues develop cells divide and differentiate concurrently. Conflicting evidence shows that cell division is either dispensable or required for formation of cell types. To determine the role of cell division in differentiation we arrested the cell cycle in zebrafish embryos using two independent approaches and profiled them at single cell resolution. We show that cell division is dispensable for differentiation of all embryonic tissues during initial cell type differentiation from early gastrulation to the end of segmentation. However in the absence of cell division differentiation slows down in some cell types and cells exhibit global stress responses. While differentiation is robust to blocking cell division the proportions of cells across cell states are not but show evidence of partial compensation. This work clarifies our understanding of the role of cell division in development and showcases the utility of combining embryo wide perturbations with single cell RNA sequencing to uncover the role of common biological processes across multiple tissues. Overall design: We arrested the cell cycle in zebrafish embryos at 6 hpf using two independent approaches: a chemical approach hydroxyurea and aphidicolin HUA and a genetic approach involving a loss of function mutation in the gene emi1 allele hi2648. We tracked differentiation dynamics using single cell RNA sequencing scRNA seq on embryos spanning 6–24 hpf for HUA treated and control embryos and at 24 hpf for emi1 mutant embryos. Overall we have collected multiple replicates for control embryos ABs at 6 8 10 14 18 21 hpf and 24 hpf for HUA treated at 8 10 14 hpf and 24 hpf and for emi1 mutants at 24 hpf. Details about the samples can be found in the supplementary file "sample information.txt"   pubmed:37546736   Perturbation experiment 2 24 hpf biological replicate 1 technical replicate 1 to 4 GSM8327216   source name:whole embryo|tissue:whole embryo|treatment:1percent DMSO control and cell cycle arrest at 6 hpf|geo loc name:missing|collection date:missing Perturbation experiment 2 24 hpf biological replicate 1 technical replicate 1 to 4 Multiple samples are pooled for each sequencing run. Raw FASTQ were prepared from Illumina bcl files in a format compatible with the inDrops.py pipeline. Each nextseq run results in 16 FASTQ files 4 lanes x 4 reads per lane. For some pooled libraries sequencing was run twice to get more UMIs per cell and hence we have deposited 16x2 = 32 raw files for those sequencing samples. The illumina library indices of different samples in a run are provided in the meta data. These can be used to demultiplex the raw file into separate libraries. The read files from an inDrops run have the following content: * R1 001.fastq.gz : contains the three prime UTR cDNA read * R2 001.fastq.gz : contains the first half of the cell barcode * R3 001.fastq.gz : contains the library index for demultiplexing libraries * R4 001.fastq.gz : contains the second half of the barcode and the UMI. All subsequent processing steps from FASTQ files to count matrixes were performed using the custom pipeline for inDrops data analysis github.com/indrops. Single cell transcriptomes were barcoded using inDrops Klein et al Cell 2015. Standard transcriptome RNA seq libraries were processed as reported in Zilionis et al. Nature Protocol 2016 using inDrops v3 protocol. The transcriptome libraries were sequenced on reads Illumina NextSeq 500. Libraries used standard Illumina sequencing primers and 61 cycles for Read1 14 cycles for Read2 8 cycles each for IndexRead1 and IndexRead2. Raw fastq files was processed using inDrops.py pipeline github.com/indrops/indrops. Sequenced reads were mapped to a zebrafish reference transcriptome built from the zebrafish GRCz10 genome assembly Assembly Accession: GCF 000002035.5 using bowtie version 1.1.143. To obtain the final counts matrix used for data analysis total count filters were applied as described in Kukreja et. al. 2024 method section "Single cell RNA seq Data preprocessing" Assembly: GRCz10 genome assembly Assembly Accession: GCF 000002035.5 Supplementary files format and content: Raw counts tsv.gz: cell ba… whole embryo Zebrafish embryos were arrested for cell cycle using two complementary approaches. S phase cell cycle arrest was induced using a cocktail of drugs – hydroxyurea Sigma Aldrich H8627 5G at 20 mM and aphidicolin Sigma Aldrich A0781 5MG at 150 µM concentration in 1% dimethyl sulfoxide DMSO in egg water. G2/M phase arrest was induced by crossing heterozygous emi1 wt/mut zebrafish to generate homozygous emi1 mut/mut embryos referred as hi2648 in the manuscript. Homozygous mutants with cell cycle arrest were screened at 24 hpf as they have very clear phenotype by this time – these embryos are smaller and have bent tails and the heterozygous mutants and wildtype are indistinguishable. Zebrafish embryos were dechorionated using 1mg/mL Pronase Sigma P5147 1G for 5 7 minutes followed by washing in egg water. Embryos were dissociated as previously described in Wagner et. al. Science 2018. Briefly embryos were dissociated in 0.5mL LoBind microcentrifuge tubes that had been precoated with 10% w/v BSA for about 15 minutes at room temperature. They were homogenized in 1XDPBS/1% w/v BSA for 6 hpf to 10 hpf embryos early time points and in 500 µL FACSmax cell dissociation solution Genlantis T200100 for 14 hpf to 24 hpf embryos late time points. Cells were then filtered through a 40µm cell strainer mesh Fisher 352340 and centrifuged in a swinging bucket rotor at 200g for 1 minute early time points or 300g for 5 minutes late time points. Cells were then washed in 1XDPBS/1%BSA using the same centrifuge settings. They were then resuspended in 1XDPBS/0.5%BSA/18% optiprep density medium Sigma D1556 250ML. Cell concentration was manually quantified using hemocytometer and adjusted to a final concentration of 100 000 cells/mL before encapsulation. Library construction as detailed in Zilionis R. et al. 2016 Nature Protocols. Single cell barcoding and sequencing was done using droplet microfluidics also described in the same paper. AB wild type strains were used for all HUA perturbation experiments. Zebrafish mutant line hi2648 a mutant for the gene emi1 was kindly gifted by Dr. Jennifer Rhodes. Embryos were developed within the ambient temperature of Zebrafish development. Time of fertilization at 28.5 C was used to stage each clutch and this was confirmed using morphological features of the embryos. All zebrafish were housed in a facility overseen by the Harvard Medical Area Standing Committee on Animals our IACUC which performs regular inspections and under which we have an approved protocol for all animal procedures. tissue:whole embryo|treatment:1percent DMSO control and cell cycle arrest at 6 hpf GSM8327216 GSM8327216: Perturbation experiment 2 24 hpf biological replicate 1 technical replicate 1 to 4; Danio rerio; OTHER GSM8327216 r1 GSM8327216 1 Zebrafish embryos were dechorionated using 1mg/mL Pronase Sigma P5147 1G for 5 7 minutes followed by washing in egg water. Embryos were dissociated as previously described in Wagner et. al. Science 2018. Briefly embryos were dissociated in 0.5mL LoBind microcentrifuge tubes that had been precoated with 10% w/v BSA for about 15 minutes at room temperature. They were homogenized in 1XDPBS/1% w/v BSA for 6 hpf to 10 hpf embryos early time points and in 500 µL FACSmax cell dissociation solution Genlantis T200100 for 14 hpf to 24 hpf embryos late time points. Cells were then filtered through a 40µm cell strainer mesh Fisher 352340 and centrifuged in a swinging bucket rotor at 200g for 1 minute early time points or 300g for 5 minutes late time points. Cells were then washed in 1XDPBS/1%BSA using the same centrifuge settings. They were then resuspended in 1XDPBS/0.5%BSA/18% optiprep density medium Sigma D1556 250ML. Cell concentration was manually quantified using hemocytometer and adjusted to a final concentration of 100 000 cells/mL before encapsulation. Library construction as detailed in Zilionis R. et al. 2016 Nature Protocols. Single cell barcoding and sequencing was done using droplet microfluidics also described in the same paper.   OTHER TRANSCRIPTOMIC other PAIRED ILLUMINA NextSeq 500   SRP513754     pert_exp2_brep1_trep1_2_3_4_S0_L004_R1_001.fastq.gz pert_exp2_brep1_trep1_2_3_4_S0_L004_R2_001.fastq.gz pert_exp2_brep1_trep1_2_3_4_S0_L004_R3_001.fastq.gz pert_exp2_brep1_trep1_2_3_4_S0_L004_R4_001.fastq.gz fastq fastq fastq fastq 11081541198.0 121775178.0 GSM8327216 r4 0:61 1:8 2:8 3:14 A:1869228214;C:1474483692;G:1435361382;T:2648837266;N:375304 61 8 8 14 1869228214 1474483692 1435361382 2648837266 375304 SRX24912667 SRS21618602 SRA1898111 Harvard University Harvard University                       B   usable mapping rate illumina nextseq unknown other trueseq sc single_cell_droplet indrops   United States 2024-06-13 Multi-stage Embryo Whole Organism All anatomical structures
32745 32745 SRR29398884 SRX24912666 SRS21618600 SRP513754 PRJNA1123686 Cell state transitions are decoupled from cell division during early embryo development [I] GSE269784 Other As tissues develop cells divide and differentiate concurrently. Conflicting evidence shows that cell division is either dispensable or required for formation of cell types. To determine the role of cell division in differentiation we arrested the cell cycle in zebrafish embryos using two independent approaches and profiled them at single cell resolution. We show that cell division is dispensable for differentiation of all embryonic tissues during initial cell type differentiation from early gastrulation to the end of segmentation. However in the absence of cell division differentiation slows down in some cell types and cells exhibit global stress responses. While differentiation is robust to blocking cell division the proportions of cells across cell states are not but show evidence of partial compensation. This work clarifies our understanding of the role of cell division in development and showcases the utility of combining embryo wide perturbations with single cell RNA sequencing to uncover the role of common biological processes across multiple tissues. Overall design: We arrested the cell cycle in zebrafish embryos at 6 hpf using two independent approaches: a chemical approach hydroxyurea and aphidicolin HUA and a genetic approach involving a loss of function mutation in the gene emi1 allele hi2648. We tracked differentiation dynamics using single cell RNA sequencing scRNA seq on embryos spanning 6–24 hpf for HUA treated and control embryos and at 24 hpf for emi1 mutant embryos. Overall we have collected multiple replicates for control embryos ABs at 6 8 10 14 18 21 hpf and 24 hpf for HUA treated at 8 10 14 hpf and 24 hpf and for emi1 mutants at 24 hpf. Details about the samples can be found in the supplementary file "sample information.txt"   pubmed:37546736   Perturbation experiment 1 6 hpf 24 hpf biological replicate 3 technical replicate 1 GSM8327215   source name:whole embryo|tissue:whole embryo|treatment:1percent DMSO control and cell cycle arrest at 6 hpf|geo loc name:missing|collection date:missing Perturbation experiment 1 6 hpf 24 hpf biological replicate 3 technical replicate 1 Multiple samples are pooled for each sequencing run. Raw FASTQ were prepared from Illumina bcl files in a format compatible with the inDrops.py pipeline. Each nextseq run results in 16 FASTQ files 4 lanes x 4 reads per lane. For some pooled libraries sequencing was run twice to get more UMIs per cell and hence we have deposited 16x2 = 32 raw files for those sequencing samples. The illumina library indices of different samples in a run are provided in the meta data. These can be used to demultiplex the raw file into separate libraries. The read files from an inDrops run have the following content: * R1 001.fastq.gz : contains the three prime UTR cDNA read * R2 001.fastq.gz : contains the first half of the cell barcode * R3 001.fastq.gz : contains the library index for demultiplexing libraries * R4 001.fastq.gz : contains the second half of the barcode and the UMI. All subsequent processing steps from FASTQ files to count matrixes were performed using the custom pipeline for inDrops data analysis github.com/indrops. Single cell transcriptomes were barcoded using inDrops Klein et al Cell 2015. Standard transcriptome RNA seq libraries were processed as reported in Zilionis et al. Nature Protocol 2016 using inDrops v3 protocol. The transcriptome libraries were sequenced on reads Illumina NextSeq 500. Libraries used standard Illumina sequencing primers and 61 cycles for Read1 14 cycles for Read2 8 cycles each for IndexRead1 and IndexRead2. Raw fastq files was processed using inDrops.py pipeline github.com/indrops/indrops. Sequenced reads were mapped to a zebrafish reference transcriptome built from the zebrafish GRCz10 genome assembly Assembly Accession: GCF 000002035.5 using bowtie version 1.1.143. To obtain the final counts matrix used for data analysis total count filters were applied as described in Kukreja et. al. 2024 method section "Single cell RNA seq Data preprocessing" Assembly: GRCz10 genome assembly Assembly Accession: GCF 000002035.5 Supplementary files format and content: Raw counts tsv.gz: cell ba… whole embryo Zebrafish embryos were arrested for cell cycle using two complementary approaches. S phase cell cycle arrest was induced using a cocktail of drugs – hydroxyurea Sigma Aldrich H8627 5G at 20 mM and aphidicolin Sigma Aldrich A0781 5MG at 150 µM concentration in 1% dimethyl sulfoxide DMSO in egg water. G2/M phase arrest was induced by crossing heterozygous emi1 wt/mut zebrafish to generate homozygous emi1 mut/mut embryos referred as hi2648 in the manuscript. Homozygous mutants with cell cycle arrest were screened at 24 hpf as they have very clear phenotype by this time – these embryos are smaller and have bent tails and the heterozygous mutants and wildtype are indistinguishable. Zebrafish embryos were dechorionated using 1mg/mL Pronase Sigma P5147 1G for 5 7 minutes followed by washing in egg water. Embryos were dissociated as previously described in Wagner et. al. Science 2018. Briefly embryos were dissociated in 0.5mL LoBind microcentrifuge tubes that had been precoated with 10% w/v BSA for about 15 minutes at room temperature. They were homogenized in 1XDPBS/1% w/v BSA for 6 hpf to 10 hpf embryos early time points and in 500 µL FACSmax cell dissociation solution Genlantis T200100 for 14 hpf to 24 hpf embryos late time points. Cells were then filtered through a 40µm cell strainer mesh Fisher 352340 and centrifuged in a swinging bucket rotor at 200g for 1 minute early time points or 300g for 5 minutes late time points. Cells were then washed in 1XDPBS/1%BSA using the same centrifuge settings. They were then resuspended in 1XDPBS/0.5%BSA/18% optiprep density medium Sigma D1556 250ML. Cell concentration was manually quantified using hemocytometer and adjusted to a final concentration of 100 000 cells/mL before encapsulation. Library construction as detailed in Zilionis R. et al. 2016 Nature Protocols. Single cell barcoding and sequencing was done using droplet microfluidics also described in the same paper. AB wild type strains were used for all HUA perturbation experiments. Zebrafish mutant line hi2648 a mutant for the gene emi1 was kindly gifted by Dr. Jennifer Rhodes. Embryos were developed within the ambient temperature of Zebrafish development. Time of fertilization at 28.5 C was used to stage each clutch and this was confirmed using morphological features of the embryos. All zebrafish were housed in a facility overseen by the Harvard Medical Area Standing Committee on Animals our IACUC which performs regular inspections and under which we have an approved protocol for all animal procedures. tissue:whole embryo|treatment:1percent DMSO control and cell cycle arrest at 6 hpf GSM8327215 GSM8327215: Perturbation experiment 1 6 hpf 24 hpf biological replicate 3 technical replicate 1; Danio rerio; OTHER GSM8327215 r1 GSM8327215 1 Zebrafish embryos were dechorionated using 1mg/mL Pronase Sigma P5147 1G for 5 7 minutes followed by washing in egg water. Embryos were dissociated as previously described in Wagner et. al. Science 2018. Briefly embryos were dissociated in 0.5mL LoBind microcentrifuge tubes that had been precoated with 10% w/v BSA for about 15 minutes at room temperature. They were homogenized in 1XDPBS/1% w/v BSA for 6 hpf to 10 hpf embryos early time points and in 500 µL FACSmax cell dissociation solution Genlantis T200100 for 14 hpf to 24 hpf embryos late time points. Cells were then filtered through a 40µm cell strainer mesh Fisher 352340 and centrifuged in a swinging bucket rotor at 200g for 1 minute early time points or 300g for 5 minutes late time points. Cells were then washed in 1XDPBS/1%BSA using the same centrifuge settings. They were then resuspended in 1XDPBS/0.5%BSA/18% optiprep density medium Sigma D1556 250ML. Cell concentration was manually quantified using hemocytometer and adjusted to a final concentration of 100 000 cells/mL before encapsulation. Library construction as detailed in Zilionis R. et al. 2016 Nature Protocols. Single cell barcoding and sequencing was done using droplet microfluidics also described in the same paper.   OTHER TRANSCRIPTOMIC other PAIRED ILLUMINA NextSeq 500   SRP513754     pert_exp1_brep3_trep1_S0_L001_R1_001.fastq.gz pert_exp1_brep3_trep1_S0_L001_R2_001.fastq.gz pert_exp1_brep3_trep1_S0_L001_R3_001.fastq.gz pert_exp1_brep3_trep1_S0_L001_R4_001.fastq.gz fastq fastq fastq fastq 9825258079.0 107969869.0 GSM8327215 r1 0:61 1:8 2:8 3:14 A:1892998816;C:1448298491;G:1287308183;T:1956393917;N:1162602 61 8 8 14 1892998816 1448298491 1287308183 1956393917 1162602 SRX24912666 SRS21618600 SRA1898111 Harvard University Harvard University                       B   usable mapping rate illumina nextseq unknown other trueseq sc single_cell_droplet indrops   United States 2024-06-13 Multi-stage Embryo Whole Organism All anatomical structures
32746 32746 SRR29398885 SRX24912666 SRS21618600 SRP513754 PRJNA1123686 Cell state transitions are decoupled from cell division during early embryo development [I] GSE269784 Other As tissues develop cells divide and differentiate concurrently. Conflicting evidence shows that cell division is either dispensable or required for formation of cell types. To determine the role of cell division in differentiation we arrested the cell cycle in zebrafish embryos using two independent approaches and profiled them at single cell resolution. We show that cell division is dispensable for differentiation of all embryonic tissues during initial cell type differentiation from early gastrulation to the end of segmentation. However in the absence of cell division differentiation slows down in some cell types and cells exhibit global stress responses. While differentiation is robust to blocking cell division the proportions of cells across cell states are not but show evidence of partial compensation. This work clarifies our understanding of the role of cell division in development and showcases the utility of combining embryo wide perturbations with single cell RNA sequencing to uncover the role of common biological processes across multiple tissues. Overall design: We arrested the cell cycle in zebrafish embryos at 6 hpf using two independent approaches: a chemical approach hydroxyurea and aphidicolin HUA and a genetic approach involving a loss of function mutation in the gene emi1 allele hi2648. We tracked differentiation dynamics using single cell RNA sequencing scRNA seq on embryos spanning 6–24 hpf for HUA treated and control embryos and at 24 hpf for emi1 mutant embryos. Overall we have collected multiple replicates for control embryos ABs at 6 8 10 14 18 21 hpf and 24 hpf for HUA treated at 8 10 14 hpf and 24 hpf and for emi1 mutants at 24 hpf. Details about the samples can be found in the supplementary file "sample information.txt"   pubmed:37546736   Perturbation experiment 1 6 hpf 24 hpf biological replicate 3 technical replicate 1 GSM8327215   source name:whole embryo|tissue:whole embryo|treatment:1percent DMSO control and cell cycle arrest at 6 hpf|geo loc name:missing|collection date:missing Perturbation experiment 1 6 hpf 24 hpf biological replicate 3 technical replicate 1 Multiple samples are pooled for each sequencing run. Raw FASTQ were prepared from Illumina bcl files in a format compatible with the inDrops.py pipeline. Each nextseq run results in 16 FASTQ files 4 lanes x 4 reads per lane. For some pooled libraries sequencing was run twice to get more UMIs per cell and hence we have deposited 16x2 = 32 raw files for those sequencing samples. The illumina library indices of different samples in a run are provided in the meta data. These can be used to demultiplex the raw file into separate libraries. The read files from an inDrops run have the following content: * R1 001.fastq.gz : contains the three prime UTR cDNA read * R2 001.fastq.gz : contains the first half of the cell barcode * R3 001.fastq.gz : contains the library index for demultiplexing libraries * R4 001.fastq.gz : contains the second half of the barcode and the UMI. All subsequent processing steps from FASTQ files to count matrixes were performed using the custom pipeline for inDrops data analysis github.com/indrops. Single cell transcriptomes were barcoded using inDrops Klein et al Cell 2015. Standard transcriptome RNA seq libraries were processed as reported in Zilionis et al. Nature Protocol 2016 using inDrops v3 protocol. The transcriptome libraries were sequenced on reads Illumina NextSeq 500. Libraries used standard Illumina sequencing primers and 61 cycles for Read1 14 cycles for Read2 8 cycles each for IndexRead1 and IndexRead2. Raw fastq files was processed using inDrops.py pipeline github.com/indrops/indrops. Sequenced reads were mapped to a zebrafish reference transcriptome built from the zebrafish GRCz10 genome assembly Assembly Accession: GCF 000002035.5 using bowtie version 1.1.143. To obtain the final counts matrix used for data analysis total count filters were applied as described in Kukreja et. al. 2024 method section "Single cell RNA seq Data preprocessing" Assembly: GRCz10 genome assembly Assembly Accession: GCF 000002035.5 Supplementary files format and content: Raw counts tsv.gz: cell ba… whole embryo Zebrafish embryos were arrested for cell cycle using two complementary approaches. S phase cell cycle arrest was induced using a cocktail of drugs – hydroxyurea Sigma Aldrich H8627 5G at 20 mM and aphidicolin Sigma Aldrich A0781 5MG at 150 µM concentration in 1% dimethyl sulfoxide DMSO in egg water. G2/M phase arrest was induced by crossing heterozygous emi1 wt/mut zebrafish to generate homozygous emi1 mut/mut embryos referred as hi2648 in the manuscript. Homozygous mutants with cell cycle arrest were screened at 24 hpf as they have very clear phenotype by this time – these embryos are smaller and have bent tails and the heterozygous mutants and wildtype are indistinguishable. Zebrafish embryos were dechorionated using 1mg/mL Pronase Sigma P5147 1G for 5 7 minutes followed by washing in egg water. Embryos were dissociated as previously described in Wagner et. al. Science 2018. Briefly embryos were dissociated in 0.5mL LoBind microcentrifuge tubes that had been precoated with 10% w/v BSA for about 15 minutes at room temperature. They were homogenized in 1XDPBS/1% w/v BSA for 6 hpf to 10 hpf embryos early time points and in 500 µL FACSmax cell dissociation solution Genlantis T200100 for 14 hpf to 24 hpf embryos late time points. Cells were then filtered through a 40µm cell strainer mesh Fisher 352340 and centrifuged in a swinging bucket rotor at 200g for 1 minute early time points or 300g for 5 minutes late time points. Cells were then washed in 1XDPBS/1%BSA using the same centrifuge settings. They were then resuspended in 1XDPBS/0.5%BSA/18% optiprep density medium Sigma D1556 250ML. Cell concentration was manually quantified using hemocytometer and adjusted to a final concentration of 100 000 cells/mL before encapsulation. Library construction as detailed in Zilionis R. et al. 2016 Nature Protocols. Single cell barcoding and sequencing was done using droplet microfluidics also described in the same paper. AB wild type strains were used for all HUA perturbation experiments. Zebrafish mutant line hi2648 a mutant for the gene emi1 was kindly gifted by Dr. Jennifer Rhodes. Embryos were developed within the ambient temperature of Zebrafish development. Time of fertilization at 28.5 C was used to stage each clutch and this was confirmed using morphological features of the embryos. All zebrafish were housed in a facility overseen by the Harvard Medical Area Standing Committee on Animals our IACUC which performs regular inspections and under which we have an approved protocol for all animal procedures. tissue:whole embryo|treatment:1percent DMSO control and cell cycle arrest at 6 hpf GSM8327215 GSM8327215: Perturbation experiment 1 6 hpf 24 hpf biological replicate 3 technical replicate 1; Danio rerio; OTHER GSM8327215 r1 GSM8327215 1 Zebrafish embryos were dechorionated using 1mg/mL Pronase Sigma P5147 1G for 5 7 minutes followed by washing in egg water. Embryos were dissociated as previously described in Wagner et. al. Science 2018. Briefly embryos were dissociated in 0.5mL LoBind microcentrifuge tubes that had been precoated with 10% w/v BSA for about 15 minutes at room temperature. They were homogenized in 1XDPBS/1% w/v BSA for 6 hpf to 10 hpf embryos early time points and in 500 µL FACSmax cell dissociation solution Genlantis T200100 for 14 hpf to 24 hpf embryos late time points. Cells were then filtered through a 40µm cell strainer mesh Fisher 352340 and centrifuged in a swinging bucket rotor at 200g for 1 minute early time points or 300g for 5 minutes late time points. Cells were then washed in 1XDPBS/1%BSA using the same centrifuge settings. They were then resuspended in 1XDPBS/0.5%BSA/18% optiprep density medium Sigma D1556 250ML. Cell concentration was manually quantified using hemocytometer and adjusted to a final concentration of 100 000 cells/mL before encapsulation. Library construction as detailed in Zilionis R. et al. 2016 Nature Protocols. Single cell barcoding and sequencing was done using droplet microfluidics also described in the same paper.   OTHER TRANSCRIPTOMIC other PAIRED ILLUMINA NextSeq 500   SRP513754     pert_exp1_brep3_trep1_S0_L002_R1_001.fastq.gz pert_exp1_brep3_trep1_S0_L002_R2_001.fastq.gz pert_exp1_brep3_trep1_S0_L002_R3_001.fastq.gz pert_exp1_brep3_trep1_S0_L002_R4_001.fastq.gz fastq fastq fastq fastq 9797467225.0 107664475.0 GSM8327215 r2 0:61 1:8 2:8 3:14 A:1884493825;C:1436463441;G:1294831461;T:1950665696;N:1078552 61 8 8 14 1884493825 1436463441 1294831461 1950665696 1078552 SRX24912666 SRS21618600 SRA1898111 Harvard University Harvard University                       B   usable mapping rate illumina nextseq unknown other trueseq sc single_cell_droplet indrops   United States 2024-06-13 Multi-stage Embryo Whole Organism All anatomical structures
32747 32747 SRR29398886 SRX24912666 SRS21618600 SRP513754 PRJNA1123686 Cell state transitions are decoupled from cell division during early embryo development [I] GSE269784 Other As tissues develop cells divide and differentiate concurrently. Conflicting evidence shows that cell division is either dispensable or required for formation of cell types. To determine the role of cell division in differentiation we arrested the cell cycle in zebrafish embryos using two independent approaches and profiled them at single cell resolution. We show that cell division is dispensable for differentiation of all embryonic tissues during initial cell type differentiation from early gastrulation to the end of segmentation. However in the absence of cell division differentiation slows down in some cell types and cells exhibit global stress responses. While differentiation is robust to blocking cell division the proportions of cells across cell states are not but show evidence of partial compensation. This work clarifies our understanding of the role of cell division in development and showcases the utility of combining embryo wide perturbations with single cell RNA sequencing to uncover the role of common biological processes across multiple tissues. Overall design: We arrested the cell cycle in zebrafish embryos at 6 hpf using two independent approaches: a chemical approach hydroxyurea and aphidicolin HUA and a genetic approach involving a loss of function mutation in the gene emi1 allele hi2648. We tracked differentiation dynamics using single cell RNA sequencing scRNA seq on embryos spanning 6–24 hpf for HUA treated and control embryos and at 24 hpf for emi1 mutant embryos. Overall we have collected multiple replicates for control embryos ABs at 6 8 10 14 18 21 hpf and 24 hpf for HUA treated at 8 10 14 hpf and 24 hpf and for emi1 mutants at 24 hpf. Details about the samples can be found in the supplementary file "sample information.txt"   pubmed:37546736   Perturbation experiment 1 6 hpf 24 hpf biological replicate 3 technical replicate 1 GSM8327215   source name:whole embryo|tissue:whole embryo|treatment:1percent DMSO control and cell cycle arrest at 6 hpf|geo loc name:missing|collection date:missing Perturbation experiment 1 6 hpf 24 hpf biological replicate 3 technical replicate 1 Multiple samples are pooled for each sequencing run. Raw FASTQ were prepared from Illumina bcl files in a format compatible with the inDrops.py pipeline. Each nextseq run results in 16 FASTQ files 4 lanes x 4 reads per lane. For some pooled libraries sequencing was run twice to get more UMIs per cell and hence we have deposited 16x2 = 32 raw files for those sequencing samples. The illumina library indices of different samples in a run are provided in the meta data. These can be used to demultiplex the raw file into separate libraries. The read files from an inDrops run have the following content: * R1 001.fastq.gz : contains the three prime UTR cDNA read * R2 001.fastq.gz : contains the first half of the cell barcode * R3 001.fastq.gz : contains the library index for demultiplexing libraries * R4 001.fastq.gz : contains the second half of the barcode and the UMI. All subsequent processing steps from FASTQ files to count matrixes were performed using the custom pipeline for inDrops data analysis github.com/indrops. Single cell transcriptomes were barcoded using inDrops Klein et al Cell 2015. Standard transcriptome RNA seq libraries were processed as reported in Zilionis et al. Nature Protocol 2016 using inDrops v3 protocol. The transcriptome libraries were sequenced on reads Illumina NextSeq 500. Libraries used standard Illumina sequencing primers and 61 cycles for Read1 14 cycles for Read2 8 cycles each for IndexRead1 and IndexRead2. Raw fastq files was processed using inDrops.py pipeline github.com/indrops/indrops. Sequenced reads were mapped to a zebrafish reference transcriptome built from the zebrafish GRCz10 genome assembly Assembly Accession: GCF 000002035.5 using bowtie version 1.1.143. To obtain the final counts matrix used for data analysis total count filters were applied as described in Kukreja et. al. 2024 method section "Single cell RNA seq Data preprocessing" Assembly: GRCz10 genome assembly Assembly Accession: GCF 000002035.5 Supplementary files format and content: Raw counts tsv.gz: cell ba… whole embryo Zebrafish embryos were arrested for cell cycle using two complementary approaches. S phase cell cycle arrest was induced using a cocktail of drugs – hydroxyurea Sigma Aldrich H8627 5G at 20 mM and aphidicolin Sigma Aldrich A0781 5MG at 150 µM concentration in 1% dimethyl sulfoxide DMSO in egg water. G2/M phase arrest was induced by crossing heterozygous emi1 wt/mut zebrafish to generate homozygous emi1 mut/mut embryos referred as hi2648 in the manuscript. Homozygous mutants with cell cycle arrest were screened at 24 hpf as they have very clear phenotype by this time – these embryos are smaller and have bent tails and the heterozygous mutants and wildtype are indistinguishable. Zebrafish embryos were dechorionated using 1mg/mL Pronase Sigma P5147 1G for 5 7 minutes followed by washing in egg water. Embryos were dissociated as previously described in Wagner et. al. Science 2018. Briefly embryos were dissociated in 0.5mL LoBind microcentrifuge tubes that had been precoated with 10% w/v BSA for about 15 minutes at room temperature. They were homogenized in 1XDPBS/1% w/v BSA for 6 hpf to 10 hpf embryos early time points and in 500 µL FACSmax cell dissociation solution Genlantis T200100 for 14 hpf to 24 hpf embryos late time points. Cells were then filtered through a 40µm cell strainer mesh Fisher 352340 and centrifuged in a swinging bucket rotor at 200g for 1 minute early time points or 300g for 5 minutes late time points. Cells were then washed in 1XDPBS/1%BSA using the same centrifuge settings. They were then resuspended in 1XDPBS/0.5%BSA/18% optiprep density medium Sigma D1556 250ML. Cell concentration was manually quantified using hemocytometer and adjusted to a final concentration of 100 000 cells/mL before encapsulation. Library construction as detailed in Zilionis R. et al. 2016 Nature Protocols. Single cell barcoding and sequencing was done using droplet microfluidics also described in the same paper. AB wild type strains were used for all HUA perturbation experiments. Zebrafish mutant line hi2648 a mutant for the gene emi1 was kindly gifted by Dr. Jennifer Rhodes. Embryos were developed within the ambient temperature of Zebrafish development. Time of fertilization at 28.5 C was used to stage each clutch and this was confirmed using morphological features of the embryos. All zebrafish were housed in a facility overseen by the Harvard Medical Area Standing Committee on Animals our IACUC which performs regular inspections and under which we have an approved protocol for all animal procedures. tissue:whole embryo|treatment:1percent DMSO control and cell cycle arrest at 6 hpf GSM8327215 GSM8327215: Perturbation experiment 1 6 hpf 24 hpf biological replicate 3 technical replicate 1; Danio rerio; OTHER GSM8327215 r1 GSM8327215 1 Zebrafish embryos were dechorionated using 1mg/mL Pronase Sigma P5147 1G for 5 7 minutes followed by washing in egg water. Embryos were dissociated as previously described in Wagner et. al. Science 2018. Briefly embryos were dissociated in 0.5mL LoBind microcentrifuge tubes that had been precoated with 10% w/v BSA for about 15 minutes at room temperature. They were homogenized in 1XDPBS/1% w/v BSA for 6 hpf to 10 hpf embryos early time points and in 500 µL FACSmax cell dissociation solution Genlantis T200100 for 14 hpf to 24 hpf embryos late time points. Cells were then filtered through a 40µm cell strainer mesh Fisher 352340 and centrifuged in a swinging bucket rotor at 200g for 1 minute early time points or 300g for 5 minutes late time points. Cells were then washed in 1XDPBS/1%BSA using the same centrifuge settings. They were then resuspended in 1XDPBS/0.5%BSA/18% optiprep density medium Sigma D1556 250ML. Cell concentration was manually quantified using hemocytometer and adjusted to a final concentration of 100 000 cells/mL before encapsulation. Library construction as detailed in Zilionis R. et al. 2016 Nature Protocols. Single cell barcoding and sequencing was done using droplet microfluidics also described in the same paper.   OTHER TRANSCRIPTOMIC other PAIRED ILLUMINA NextSeq 500   SRP513754     pert_exp1_brep3_trep1_S0_L003_R1_001.fastq.gz pert_exp1_brep3_trep1_S0_L003_R2_001.fastq.gz pert_exp1_brep3_trep1_S0_L003_R3_001.fastq.gz pert_exp1_brep3_trep1_S0_L003_R4_001.fastq.gz fastq fastq fastq fastq 9945377715.0 109289865.0 GSM8327215 r3 0:61 1:8 2:8 3:14 A:1919403458;C:1456796407;G:1307295125;T:1982126987;N:1059788 61 8 8 14 1919403458 1456796407 1307295125 1982126987 1059788 SRX24912666 SRS21618600 SRA1898111 Harvard University Harvard University                       B   usable mapping rate illumina nextseq unknown other trueseq sc single_cell_droplet indrops   United States 2024-06-13 Multi-stage Embryo Whole Organism All anatomical structures
32748 32748 SRR29398887 SRX24912666 SRS21618600 SRP513754 PRJNA1123686 Cell state transitions are decoupled from cell division during early embryo development [I] GSE269784 Other As tissues develop cells divide and differentiate concurrently. Conflicting evidence shows that cell division is either dispensable or required for formation of cell types. To determine the role of cell division in differentiation we arrested the cell cycle in zebrafish embryos using two independent approaches and profiled them at single cell resolution. We show that cell division is dispensable for differentiation of all embryonic tissues during initial cell type differentiation from early gastrulation to the end of segmentation. However in the absence of cell division differentiation slows down in some cell types and cells exhibit global stress responses. While differentiation is robust to blocking cell division the proportions of cells across cell states are not but show evidence of partial compensation. This work clarifies our understanding of the role of cell division in development and showcases the utility of combining embryo wide perturbations with single cell RNA sequencing to uncover the role of common biological processes across multiple tissues. Overall design: We arrested the cell cycle in zebrafish embryos at 6 hpf using two independent approaches: a chemical approach hydroxyurea and aphidicolin HUA and a genetic approach involving a loss of function mutation in the gene emi1 allele hi2648. We tracked differentiation dynamics using single cell RNA sequencing scRNA seq on embryos spanning 6–24 hpf for HUA treated and control embryos and at 24 hpf for emi1 mutant embryos. Overall we have collected multiple replicates for control embryos ABs at 6 8 10 14 18 21 hpf and 24 hpf for HUA treated at 8 10 14 hpf and 24 hpf and for emi1 mutants at 24 hpf. Details about the samples can be found in the supplementary file "sample information.txt"   pubmed:37546736   Perturbation experiment 1 6 hpf 24 hpf biological replicate 3 technical replicate 1 GSM8327215   source name:whole embryo|tissue:whole embryo|treatment:1percent DMSO control and cell cycle arrest at 6 hpf|geo loc name:missing|collection date:missing Perturbation experiment 1 6 hpf 24 hpf biological replicate 3 technical replicate 1 Multiple samples are pooled for each sequencing run. Raw FASTQ were prepared from Illumina bcl files in a format compatible with the inDrops.py pipeline. Each nextseq run results in 16 FASTQ files 4 lanes x 4 reads per lane. For some pooled libraries sequencing was run twice to get more UMIs per cell and hence we have deposited 16x2 = 32 raw files for those sequencing samples. The illumina library indices of different samples in a run are provided in the meta data. These can be used to demultiplex the raw file into separate libraries. The read files from an inDrops run have the following content: * R1 001.fastq.gz : contains the three prime UTR cDNA read * R2 001.fastq.gz : contains the first half of the cell barcode * R3 001.fastq.gz : contains the library index for demultiplexing libraries * R4 001.fastq.gz : contains the second half of the barcode and the UMI. All subsequent processing steps from FASTQ files to count matrixes were performed using the custom pipeline for inDrops data analysis github.com/indrops. Single cell transcriptomes were barcoded using inDrops Klein et al Cell 2015. Standard transcriptome RNA seq libraries were processed as reported in Zilionis et al. Nature Protocol 2016 using inDrops v3 protocol. The transcriptome libraries were sequenced on reads Illumina NextSeq 500. Libraries used standard Illumina sequencing primers and 61 cycles for Read1 14 cycles for Read2 8 cycles each for IndexRead1 and IndexRead2. Raw fastq files was processed using inDrops.py pipeline github.com/indrops/indrops. Sequenced reads were mapped to a zebrafish reference transcriptome built from the zebrafish GRCz10 genome assembly Assembly Accession: GCF 000002035.5 using bowtie version 1.1.143. To obtain the final counts matrix used for data analysis total count filters were applied as described in Kukreja et. al. 2024 method section "Single cell RNA seq Data preprocessing" Assembly: GRCz10 genome assembly Assembly Accession: GCF 000002035.5 Supplementary files format and content: Raw counts tsv.gz: cell ba… whole embryo Zebrafish embryos were arrested for cell cycle using two complementary approaches. S phase cell cycle arrest was induced using a cocktail of drugs – hydroxyurea Sigma Aldrich H8627 5G at 20 mM and aphidicolin Sigma Aldrich A0781 5MG at 150 µM concentration in 1% dimethyl sulfoxide DMSO in egg water. G2/M phase arrest was induced by crossing heterozygous emi1 wt/mut zebrafish to generate homozygous emi1 mut/mut embryos referred as hi2648 in the manuscript. Homozygous mutants with cell cycle arrest were screened at 24 hpf as they have very clear phenotype by this time – these embryos are smaller and have bent tails and the heterozygous mutants and wildtype are indistinguishable. Zebrafish embryos were dechorionated using 1mg/mL Pronase Sigma P5147 1G for 5 7 minutes followed by washing in egg water. Embryos were dissociated as previously described in Wagner et. al. Science 2018. Briefly embryos were dissociated in 0.5mL LoBind microcentrifuge tubes that had been precoated with 10% w/v BSA for about 15 minutes at room temperature. They were homogenized in 1XDPBS/1% w/v BSA for 6 hpf to 10 hpf embryos early time points and in 500 µL FACSmax cell dissociation solution Genlantis T200100 for 14 hpf to 24 hpf embryos late time points. Cells were then filtered through a 40µm cell strainer mesh Fisher 352340 and centrifuged in a swinging bucket rotor at 200g for 1 minute early time points or 300g for 5 minutes late time points. Cells were then washed in 1XDPBS/1%BSA using the same centrifuge settings. They were then resuspended in 1XDPBS/0.5%BSA/18% optiprep density medium Sigma D1556 250ML. Cell concentration was manually quantified using hemocytometer and adjusted to a final concentration of 100 000 cells/mL before encapsulation. Library construction as detailed in Zilionis R. et al. 2016 Nature Protocols. Single cell barcoding and sequencing was done using droplet microfluidics also described in the same paper. AB wild type strains were used for all HUA perturbation experiments. Zebrafish mutant line hi2648 a mutant for the gene emi1 was kindly gifted by Dr. Jennifer Rhodes. Embryos were developed within the ambient temperature of Zebrafish development. Time of fertilization at 28.5 C was used to stage each clutch and this was confirmed using morphological features of the embryos. All zebrafish were housed in a facility overseen by the Harvard Medical Area Standing Committee on Animals our IACUC which performs regular inspections and under which we have an approved protocol for all animal procedures. tissue:whole embryo|treatment:1percent DMSO control and cell cycle arrest at 6 hpf GSM8327215 GSM8327215: Perturbation experiment 1 6 hpf 24 hpf biological replicate 3 technical replicate 1; Danio rerio; OTHER GSM8327215 r1 GSM8327215 1 Zebrafish embryos were dechorionated using 1mg/mL Pronase Sigma P5147 1G for 5 7 minutes followed by washing in egg water. Embryos were dissociated as previously described in Wagner et. al. Science 2018. Briefly embryos were dissociated in 0.5mL LoBind microcentrifuge tubes that had been precoated with 10% w/v BSA for about 15 minutes at room temperature. They were homogenized in 1XDPBS/1% w/v BSA for 6 hpf to 10 hpf embryos early time points and in 500 µL FACSmax cell dissociation solution Genlantis T200100 for 14 hpf to 24 hpf embryos late time points. Cells were then filtered through a 40µm cell strainer mesh Fisher 352340 and centrifuged in a swinging bucket rotor at 200g for 1 minute early time points or 300g for 5 minutes late time points. Cells were then washed in 1XDPBS/1%BSA using the same centrifuge settings. They were then resuspended in 1XDPBS/0.5%BSA/18% optiprep density medium Sigma D1556 250ML. Cell concentration was manually quantified using hemocytometer and adjusted to a final concentration of 100 000 cells/mL before encapsulation. Library construction as detailed in Zilionis R. et al. 2016 Nature Protocols. Single cell barcoding and sequencing was done using droplet microfluidics also described in the same paper.   OTHER TRANSCRIPTOMIC other PAIRED ILLUMINA NextSeq 500   SRP513754     pert_exp1_brep3_trep1_S0_L004_R1_001.fastq.gz pert_exp1_brep3_trep1_S0_L004_R2_001.fastq.gz pert_exp1_brep3_trep1_S0_L004_R3_001.fastq.gz pert_exp1_brep3_trep1_S0_L004_R4_001.fastq.gz fastq fastq fastq fastq 9799158005.0 107683055.0 GSM8327215 r4 0:61 1:8 2:8 3:14 A:1887332404;C:1443564157;G:1288767599;T:1948048686;N:953509 61 8 8 14 1887332404 1443564157 1288767599 1948048686 953509 SRX24912666 SRS21618600 SRA1898111 Harvard University Harvard University                       B   usable mapping rate illumina nextseq unknown other trueseq sc single_cell_droplet indrops   United States 2024-06-13 Multi-stage Embryo Whole Organism All anatomical structures
32749 32749 SRR29398888 SRX24912665 SRS21618599 SRP513754 PRJNA1123686 Cell state transitions are decoupled from cell division during early embryo development [I] GSE269784 Other As tissues develop cells divide and differentiate concurrently. Conflicting evidence shows that cell division is either dispensable or required for formation of cell types. To determine the role of cell division in differentiation we arrested the cell cycle in zebrafish embryos using two independent approaches and profiled them at single cell resolution. We show that cell division is dispensable for differentiation of all embryonic tissues during initial cell type differentiation from early gastrulation to the end of segmentation. However in the absence of cell division differentiation slows down in some cell types and cells exhibit global stress responses. While differentiation is robust to blocking cell division the proportions of cells across cell states are not but show evidence of partial compensation. This work clarifies our understanding of the role of cell division in development and showcases the utility of combining embryo wide perturbations with single cell RNA sequencing to uncover the role of common biological processes across multiple tissues. Overall design: We arrested the cell cycle in zebrafish embryos at 6 hpf using two independent approaches: a chemical approach hydroxyurea and aphidicolin HUA and a genetic approach involving a loss of function mutation in the gene emi1 allele hi2648. We tracked differentiation dynamics using single cell RNA sequencing scRNA seq on embryos spanning 6–24 hpf for HUA treated and control embryos and at 24 hpf for emi1 mutant embryos. Overall we have collected multiple replicates for control embryos ABs at 6 8 10 14 18 21 hpf and 24 hpf for HUA treated at 8 10 14 hpf and 24 hpf and for emi1 mutants at 24 hpf. Details about the samples can be found in the supplementary file "sample information.txt"   pubmed:37546736   Perturbation experiment 1 6 hpf 14 hpf biological replicate 1 technical replicate 1 GSM8327213   source name:whole embryo|tissue:whole embryo|treatment:1percent DMSO control and cell cycle arrest at 6 hpf|geo loc name:missing|collection date:missing Perturbation experiment 1 6 hpf 14 hpf biological replicate 1 technical replicate 1 Multiple samples are pooled for each sequencing run. Raw FASTQ were prepared from Illumina bcl files in a format compatible with the inDrops.py pipeline. Each nextseq run results in 16 FASTQ files 4 lanes x 4 reads per lane. For some pooled libraries sequencing was run twice to get more UMIs per cell and hence we have deposited 16x2 = 32 raw files for those sequencing samples. The illumina library indices of different samples in a run are provided in the meta data. These can be used to demultiplex the raw file into separate libraries. The read files from an inDrops run have the following content: * R1 001.fastq.gz : contains the three prime UTR cDNA read * R2 001.fastq.gz : contains the first half of the cell barcode * R3 001.fastq.gz : contains the library index for demultiplexing libraries * R4 001.fastq.gz : contains the second half of the barcode and the UMI. All subsequent processing steps from FASTQ files to count matrixes were performed using the custom pipeline for inDrops data analysis github.com/indrops. Single cell transcriptomes were barcoded using inDrops Klein et al Cell 2015. Standard transcriptome RNA seq libraries were processed as reported in Zilionis et al. Nature Protocol 2016 using inDrops v3 protocol. The transcriptome libraries were sequenced on reads Illumina NextSeq 500. Libraries used standard Illumina sequencing primers and 61 cycles for Read1 14 cycles for Read2 8 cycles each for IndexRead1 and IndexRead2. Raw fastq files was processed using inDrops.py pipeline github.com/indrops/indrops. Sequenced reads were mapped to a zebrafish reference transcriptome built from the zebrafish GRCz10 genome assembly Assembly Accession: GCF 000002035.5 using bowtie version 1.1.143. To obtain the final counts matrix used for data analysis total count filters were applied as described in Kukreja et. al. 2024 method section "Single cell RNA seq Data preprocessing" Assembly: GRCz10 genome assembly Assembly Accession: GCF 000002035.5 Supplementary files format and content: Raw counts tsv.gz: cell ba… whole embryo Zebrafish embryos were arrested for cell cycle using two complementary approaches. S phase cell cycle arrest was induced using a cocktail of drugs – hydroxyurea Sigma Aldrich H8627 5G at 20 mM and aphidicolin Sigma Aldrich A0781 5MG at 150 µM concentration in 1% dimethyl sulfoxide DMSO in egg water. G2/M phase arrest was induced by crossing heterozygous emi1 wt/mut zebrafish to generate homozygous emi1 mut/mut embryos referred as hi2648 in the manuscript. Homozygous mutants with cell cycle arrest were screened at 24 hpf as they have very clear phenotype by this time – these embryos are smaller and have bent tails and the heterozygous mutants and wildtype are indistinguishable. Zebrafish embryos were dechorionated using 1mg/mL Pronase Sigma P5147 1G for 5 7 minutes followed by washing in egg water. Embryos were dissociated as previously described in Wagner et. al. Science 2018. Briefly embryos were dissociated in 0.5mL LoBind microcentrifuge tubes that had been precoated with 10% w/v BSA for about 15 minutes at room temperature. They were homogenized in 1XDPBS/1% w/v BSA for 6 hpf to 10 hpf embryos early time points and in 500 µL FACSmax cell dissociation solution Genlantis T200100 for 14 hpf to 24 hpf embryos late time points. Cells were then filtered through a 40µm cell strainer mesh Fisher 352340 and centrifuged in a swinging bucket rotor at 200g for 1 minute early time points or 300g for 5 minutes late time points. Cells were then washed in 1XDPBS/1%BSA using the same centrifuge settings. They were then resuspended in 1XDPBS/0.5%BSA/18% optiprep density medium Sigma D1556 250ML. Cell concentration was manually quantified using hemocytometer and adjusted to a final concentration of 100 000 cells/mL before encapsulation. Library construction as detailed in Zilionis R. et al. 2016 Nature Protocols. Single cell barcoding and sequencing was done using droplet microfluidics also described in the same paper. AB wild type strains were used for all HUA perturbation experiments. Zebrafish mutant line hi2648 a mutant for the gene emi1 was kindly gifted by Dr. Jennifer Rhodes. Embryos were developed within the ambient temperature of Zebrafish development. Time of fertilization at 28.5 C was used to stage each clutch and this was confirmed using morphological features of the embryos. All zebrafish were housed in a facility overseen by the Harvard Medical Area Standing Committee on Animals our IACUC which performs regular inspections and under which we have an approved protocol for all animal procedures. tissue:whole embryo|treatment:1percent DMSO control and cell cycle arrest at 6 hpf GSM8327213 GSM8327213: Perturbation experiment 1 6 hpf 14 hpf biological replicate 1 technical replicate 1; Danio rerio; OTHER GSM8327213 r1 GSM8327213 1 Zebrafish embryos were dechorionated using 1mg/mL Pronase Sigma P5147 1G for 5 7 minutes followed by washing in egg water. Embryos were dissociated as previously described in Wagner et. al. Science 2018. Briefly embryos were dissociated in 0.5mL LoBind microcentrifuge tubes that had been precoated with 10% w/v BSA for about 15 minutes at room temperature. They were homogenized in 1XDPBS/1% w/v BSA for 6 hpf to 10 hpf embryos early time points and in 500 µL FACSmax cell dissociation solution Genlantis T200100 for 14 hpf to 24 hpf embryos late time points. Cells were then filtered through a 40µm cell strainer mesh Fisher 352340 and centrifuged in a swinging bucket rotor at 200g for 1 minute early time points or 300g for 5 minutes late time points. Cells were then washed in 1XDPBS/1%BSA using the same centrifuge settings. They were then resuspended in 1XDPBS/0.5%BSA/18% optiprep density medium Sigma D1556 250ML. Cell concentration was manually quantified using hemocytometer and adjusted to a final concentration of 100 000 cells/mL before encapsulation. Library construction as detailed in Zilionis R. et al. 2016 Nature Protocols. Single cell barcoding and sequencing was done using droplet microfluidics also described in the same paper.   OTHER TRANSCRIPTOMIC other PAIRED ILLUMINA NextSeq 500   SRP513754     pert_exp1_brep1_trep1_S0_L001_R1_001_run1.fastq.gz pert_exp1_brep1_trep1_S0_L001_R2_001_run1.fastq.gz pert_exp1_brep1_trep1_S0_L001_R3_001_run1.fastq.gz pert_exp1_brep1_trep1_S0_L001_R4_001_run1.fastq.gz fastq fastq fastq fastq 5753105540.0 63220940.0 GSM8327213 r1 0:61 1:8 2:8 3:14 A:1174237214;C:759555395;G:705449866;T:1217212379;N:22486 61 8 8 14 1174237214 759555395 705449866 1217212379 22486 SRX24912665 SRS21618599 SRA1898111 Harvard University Harvard University                       B   usable mapping rate illumina nextseq unknown other trueseq sc single_cell_droplet indrops   United States 2024-06-13 Multi-stage Embryo Whole Organism All anatomical structures
32750 32750 SRR29398889 SRX24912665 SRS21618599 SRP513754 PRJNA1123686 Cell state transitions are decoupled from cell division during early embryo development [I] GSE269784 Other As tissues develop cells divide and differentiate concurrently. Conflicting evidence shows that cell division is either dispensable or required for formation of cell types. To determine the role of cell division in differentiation we arrested the cell cycle in zebrafish embryos using two independent approaches and profiled them at single cell resolution. We show that cell division is dispensable for differentiation of all embryonic tissues during initial cell type differentiation from early gastrulation to the end of segmentation. However in the absence of cell division differentiation slows down in some cell types and cells exhibit global stress responses. While differentiation is robust to blocking cell division the proportions of cells across cell states are not but show evidence of partial compensation. This work clarifies our understanding of the role of cell division in development and showcases the utility of combining embryo wide perturbations with single cell RNA sequencing to uncover the role of common biological processes across multiple tissues. Overall design: We arrested the cell cycle in zebrafish embryos at 6 hpf using two independent approaches: a chemical approach hydroxyurea and aphidicolin HUA and a genetic approach involving a loss of function mutation in the gene emi1 allele hi2648. We tracked differentiation dynamics using single cell RNA sequencing scRNA seq on embryos spanning 6–24 hpf for HUA treated and control embryos and at 24 hpf for emi1 mutant embryos. Overall we have collected multiple replicates for control embryos ABs at 6 8 10 14 18 21 hpf and 24 hpf for HUA treated at 8 10 14 hpf and 24 hpf and for emi1 mutants at 24 hpf. Details about the samples can be found in the supplementary file "sample information.txt"   pubmed:37546736   Perturbation experiment 1 6 hpf 14 hpf biological replicate 1 technical replicate 1 GSM8327213   source name:whole embryo|tissue:whole embryo|treatment:1percent DMSO control and cell cycle arrest at 6 hpf|geo loc name:missing|collection date:missing Perturbation experiment 1 6 hpf 14 hpf biological replicate 1 technical replicate 1 Multiple samples are pooled for each sequencing run. Raw FASTQ were prepared from Illumina bcl files in a format compatible with the inDrops.py pipeline. Each nextseq run results in 16 FASTQ files 4 lanes x 4 reads per lane. For some pooled libraries sequencing was run twice to get more UMIs per cell and hence we have deposited 16x2 = 32 raw files for those sequencing samples. The illumina library indices of different samples in a run are provided in the meta data. These can be used to demultiplex the raw file into separate libraries. The read files from an inDrops run have the following content: * R1 001.fastq.gz : contains the three prime UTR cDNA read * R2 001.fastq.gz : contains the first half of the cell barcode * R3 001.fastq.gz : contains the library index for demultiplexing libraries * R4 001.fastq.gz : contains the second half of the barcode and the UMI. All subsequent processing steps from FASTQ files to count matrixes were performed using the custom pipeline for inDrops data analysis github.com/indrops. Single cell transcriptomes were barcoded using inDrops Klein et al Cell 2015. Standard transcriptome RNA seq libraries were processed as reported in Zilionis et al. Nature Protocol 2016 using inDrops v3 protocol. The transcriptome libraries were sequenced on reads Illumina NextSeq 500. Libraries used standard Illumina sequencing primers and 61 cycles for Read1 14 cycles for Read2 8 cycles each for IndexRead1 and IndexRead2. Raw fastq files was processed using inDrops.py pipeline github.com/indrops/indrops. Sequenced reads were mapped to a zebrafish reference transcriptome built from the zebrafish GRCz10 genome assembly Assembly Accession: GCF 000002035.5 using bowtie version 1.1.143. To obtain the final counts matrix used for data analysis total count filters were applied as described in Kukreja et. al. 2024 method section "Single cell RNA seq Data preprocessing" Assembly: GRCz10 genome assembly Assembly Accession: GCF 000002035.5 Supplementary files format and content: Raw counts tsv.gz: cell ba… whole embryo Zebrafish embryos were arrested for cell cycle using two complementary approaches. S phase cell cycle arrest was induced using a cocktail of drugs – hydroxyurea Sigma Aldrich H8627 5G at 20 mM and aphidicolin Sigma Aldrich A0781 5MG at 150 µM concentration in 1% dimethyl sulfoxide DMSO in egg water. G2/M phase arrest was induced by crossing heterozygous emi1 wt/mut zebrafish to generate homozygous emi1 mut/mut embryos referred as hi2648 in the manuscript. Homozygous mutants with cell cycle arrest were screened at 24 hpf as they have very clear phenotype by this time – these embryos are smaller and have bent tails and the heterozygous mutants and wildtype are indistinguishable. Zebrafish embryos were dechorionated using 1mg/mL Pronase Sigma P5147 1G for 5 7 minutes followed by washing in egg water. Embryos were dissociated as previously described in Wagner et. al. Science 2018. Briefly embryos were dissociated in 0.5mL LoBind microcentrifuge tubes that had been precoated with 10% w/v BSA for about 15 minutes at room temperature. They were homogenized in 1XDPBS/1% w/v BSA for 6 hpf to 10 hpf embryos early time points and in 500 µL FACSmax cell dissociation solution Genlantis T200100 for 14 hpf to 24 hpf embryos late time points. Cells were then filtered through a 40µm cell strainer mesh Fisher 352340 and centrifuged in a swinging bucket rotor at 200g for 1 minute early time points or 300g for 5 minutes late time points. Cells were then washed in 1XDPBS/1%BSA using the same centrifuge settings. They were then resuspended in 1XDPBS/0.5%BSA/18% optiprep density medium Sigma D1556 250ML. Cell concentration was manually quantified using hemocytometer and adjusted to a final concentration of 100 000 cells/mL before encapsulation. Library construction as detailed in Zilionis R. et al. 2016 Nature Protocols. Single cell barcoding and sequencing was done using droplet microfluidics also described in the same paper. AB wild type strains were used for all HUA perturbation experiments. Zebrafish mutant line hi2648 a mutant for the gene emi1 was kindly gifted by Dr. Jennifer Rhodes. Embryos were developed within the ambient temperature of Zebrafish development. Time of fertilization at 28.5 C was used to stage each clutch and this was confirmed using morphological features of the embryos. All zebrafish were housed in a facility overseen by the Harvard Medical Area Standing Committee on Animals our IACUC which performs regular inspections and under which we have an approved protocol for all animal procedures. tissue:whole embryo|treatment:1percent DMSO control and cell cycle arrest at 6 hpf GSM8327213 GSM8327213: Perturbation experiment 1 6 hpf 14 hpf biological replicate 1 technical replicate 1; Danio rerio; OTHER GSM8327213 r1 GSM8327213 1 Zebrafish embryos were dechorionated using 1mg/mL Pronase Sigma P5147 1G for 5 7 minutes followed by washing in egg water. Embryos were dissociated as previously described in Wagner et. al. Science 2018. Briefly embryos were dissociated in 0.5mL LoBind microcentrifuge tubes that had been precoated with 10% w/v BSA for about 15 minutes at room temperature. They were homogenized in 1XDPBS/1% w/v BSA for 6 hpf to 10 hpf embryos early time points and in 500 µL FACSmax cell dissociation solution Genlantis T200100 for 14 hpf to 24 hpf embryos late time points. Cells were then filtered through a 40µm cell strainer mesh Fisher 352340 and centrifuged in a swinging bucket rotor at 200g for 1 minute early time points or 300g for 5 minutes late time points. Cells were then washed in 1XDPBS/1%BSA using the same centrifuge settings. They were then resuspended in 1XDPBS/0.5%BSA/18% optiprep density medium Sigma D1556 250ML. Cell concentration was manually quantified using hemocytometer and adjusted to a final concentration of 100 000 cells/mL before encapsulation. Library construction as detailed in Zilionis R. et al. 2016 Nature Protocols. Single cell barcoding and sequencing was done using droplet microfluidics also described in the same paper.   OTHER TRANSCRIPTOMIC other PAIRED ILLUMINA NextSeq 500   SRP513754     pert_exp1_brep1_trep1_S0_L002_R1_001_run1.fastq.gz pert_exp1_brep1_trep1_S0_L002_R2_001_run1.fastq.gz pert_exp1_brep1_trep1_S0_L002_R3_001_run1.fastq.gz pert_exp1_brep1_trep1_S0_L002_R4_001_run1.fastq.gz fastq fastq fastq fastq 5588689379.0 61414169.0 GSM8327213 r2 0:61 1:8 2:8 3:14 A:1135353331;C:739291995;G:689386853;T:1182212845;N:19285 61 8 8 14 1135353331 739291995 689386853 1182212845 19285 SRX24912665 SRS21618599 SRA1898111 Harvard University Harvard University                       B   usable mapping rate illumina nextseq unknown other trueseq sc single_cell_droplet indrops   United States 2024-06-13 Multi-stage Embryo Whole Organism All anatomical structures
32751 32751 SRR29398890 SRX24912665 SRS21618599 SRP513754 PRJNA1123686 Cell state transitions are decoupled from cell division during early embryo development [I] GSE269784 Other As tissues develop cells divide and differentiate concurrently. Conflicting evidence shows that cell division is either dispensable or required for formation of cell types. To determine the role of cell division in differentiation we arrested the cell cycle in zebrafish embryos using two independent approaches and profiled them at single cell resolution. We show that cell division is dispensable for differentiation of all embryonic tissues during initial cell type differentiation from early gastrulation to the end of segmentation. However in the absence of cell division differentiation slows down in some cell types and cells exhibit global stress responses. While differentiation is robust to blocking cell division the proportions of cells across cell states are not but show evidence of partial compensation. This work clarifies our understanding of the role of cell division in development and showcases the utility of combining embryo wide perturbations with single cell RNA sequencing to uncover the role of common biological processes across multiple tissues. Overall design: We arrested the cell cycle in zebrafish embryos at 6 hpf using two independent approaches: a chemical approach hydroxyurea and aphidicolin HUA and a genetic approach involving a loss of function mutation in the gene emi1 allele hi2648. We tracked differentiation dynamics using single cell RNA sequencing scRNA seq on embryos spanning 6–24 hpf for HUA treated and control embryos and at 24 hpf for emi1 mutant embryos. Overall we have collected multiple replicates for control embryos ABs at 6 8 10 14 18 21 hpf and 24 hpf for HUA treated at 8 10 14 hpf and 24 hpf and for emi1 mutants at 24 hpf. Details about the samples can be found in the supplementary file "sample information.txt"   pubmed:37546736   Perturbation experiment 1 6 hpf 14 hpf biological replicate 1 technical replicate 1 GSM8327213   source name:whole embryo|tissue:whole embryo|treatment:1percent DMSO control and cell cycle arrest at 6 hpf|geo loc name:missing|collection date:missing Perturbation experiment 1 6 hpf 14 hpf biological replicate 1 technical replicate 1 Multiple samples are pooled for each sequencing run. Raw FASTQ were prepared from Illumina bcl files in a format compatible with the inDrops.py pipeline. Each nextseq run results in 16 FASTQ files 4 lanes x 4 reads per lane. For some pooled libraries sequencing was run twice to get more UMIs per cell and hence we have deposited 16x2 = 32 raw files for those sequencing samples. The illumina library indices of different samples in a run are provided in the meta data. These can be used to demultiplex the raw file into separate libraries. The read files from an inDrops run have the following content: * R1 001.fastq.gz : contains the three prime UTR cDNA read * R2 001.fastq.gz : contains the first half of the cell barcode * R3 001.fastq.gz : contains the library index for demultiplexing libraries * R4 001.fastq.gz : contains the second half of the barcode and the UMI. All subsequent processing steps from FASTQ files to count matrixes were performed using the custom pipeline for inDrops data analysis github.com/indrops. Single cell transcriptomes were barcoded using inDrops Klein et al Cell 2015. Standard transcriptome RNA seq libraries were processed as reported in Zilionis et al. Nature Protocol 2016 using inDrops v3 protocol. The transcriptome libraries were sequenced on reads Illumina NextSeq 500. Libraries used standard Illumina sequencing primers and 61 cycles for Read1 14 cycles for Read2 8 cycles each for IndexRead1 and IndexRead2. Raw fastq files was processed using inDrops.py pipeline github.com/indrops/indrops. Sequenced reads were mapped to a zebrafish reference transcriptome built from the zebrafish GRCz10 genome assembly Assembly Accession: GCF 000002035.5 using bowtie version 1.1.143. To obtain the final counts matrix used for data analysis total count filters were applied as described in Kukreja et. al. 2024 method section "Single cell RNA seq Data preprocessing" Assembly: GRCz10 genome assembly Assembly Accession: GCF 000002035.5 Supplementary files format and content: Raw counts tsv.gz: cell ba… whole embryo Zebrafish embryos were arrested for cell cycle using two complementary approaches. S phase cell cycle arrest was induced using a cocktail of drugs – hydroxyurea Sigma Aldrich H8627 5G at 20 mM and aphidicolin Sigma Aldrich A0781 5MG at 150 µM concentration in 1% dimethyl sulfoxide DMSO in egg water. G2/M phase arrest was induced by crossing heterozygous emi1 wt/mut zebrafish to generate homozygous emi1 mut/mut embryos referred as hi2648 in the manuscript. Homozygous mutants with cell cycle arrest were screened at 24 hpf as they have very clear phenotype by this time – these embryos are smaller and have bent tails and the heterozygous mutants and wildtype are indistinguishable. Zebrafish embryos were dechorionated using 1mg/mL Pronase Sigma P5147 1G for 5 7 minutes followed by washing in egg water. Embryos were dissociated as previously described in Wagner et. al. Science 2018. Briefly embryos were dissociated in 0.5mL LoBind microcentrifuge tubes that had been precoated with 10% w/v BSA for about 15 minutes at room temperature. They were homogenized in 1XDPBS/1% w/v BSA for 6 hpf to 10 hpf embryos early time points and in 500 µL FACSmax cell dissociation solution Genlantis T200100 for 14 hpf to 24 hpf embryos late time points. Cells were then filtered through a 40µm cell strainer mesh Fisher 352340 and centrifuged in a swinging bucket rotor at 200g for 1 minute early time points or 300g for 5 minutes late time points. Cells were then washed in 1XDPBS/1%BSA using the same centrifuge settings. They were then resuspended in 1XDPBS/0.5%BSA/18% optiprep density medium Sigma D1556 250ML. Cell concentration was manually quantified using hemocytometer and adjusted to a final concentration of 100 000 cells/mL before encapsulation. Library construction as detailed in Zilionis R. et al. 2016 Nature Protocols. Single cell barcoding and sequencing was done using droplet microfluidics also described in the same paper. AB wild type strains were used for all HUA perturbation experiments. Zebrafish mutant line hi2648 a mutant for the gene emi1 was kindly gifted by Dr. Jennifer Rhodes. Embryos were developed within the ambient temperature of Zebrafish development. Time of fertilization at 28.5 C was used to stage each clutch and this was confirmed using morphological features of the embryos. All zebrafish were housed in a facility overseen by the Harvard Medical Area Standing Committee on Animals our IACUC which performs regular inspections and under which we have an approved protocol for all animal procedures. tissue:whole embryo|treatment:1percent DMSO control and cell cycle arrest at 6 hpf GSM8327213 GSM8327213: Perturbation experiment 1 6 hpf 14 hpf biological replicate 1 technical replicate 1; Danio rerio; OTHER GSM8327213 r1 GSM8327213 1 Zebrafish embryos were dechorionated using 1mg/mL Pronase Sigma P5147 1G for 5 7 minutes followed by washing in egg water. Embryos were dissociated as previously described in Wagner et. al. Science 2018. Briefly embryos were dissociated in 0.5mL LoBind microcentrifuge tubes that had been precoated with 10% w/v BSA for about 15 minutes at room temperature. They were homogenized in 1XDPBS/1% w/v BSA for 6 hpf to 10 hpf embryos early time points and in 500 µL FACSmax cell dissociation solution Genlantis T200100 for 14 hpf to 24 hpf embryos late time points. Cells were then filtered through a 40µm cell strainer mesh Fisher 352340 and centrifuged in a swinging bucket rotor at 200g for 1 minute early time points or 300g for 5 minutes late time points. Cells were then washed in 1XDPBS/1%BSA using the same centrifuge settings. They were then resuspended in 1XDPBS/0.5%BSA/18% optiprep density medium Sigma D1556 250ML. Cell concentration was manually quantified using hemocytometer and adjusted to a final concentration of 100 000 cells/mL before encapsulation. Library construction as detailed in Zilionis R. et al. 2016 Nature Protocols. Single cell barcoding and sequencing was done using droplet microfluidics also described in the same paper.   OTHER TRANSCRIPTOMIC other PAIRED ILLUMINA NextSeq 500   SRP513754     pert_exp1_brep1_trep1_S0_L003_R1_001_run1.fastq.gz pert_exp1_brep1_trep1_S0_L003_R2_001_run1.fastq.gz pert_exp1_brep1_trep1_S0_L003_R3_001_run1.fastq.gz pert_exp1_brep1_trep1_S0_L003_R4_001_run1.fastq.gz fastq fastq fastq fastq 5731949769.0 62988459.0 GSM8327213 r3 0:61 1:8 2:8 3:14 A:1167937533;C:754846871;G:708985526;T:1210491951;N:34118 61 8 8 14 1167937533 754846871 708985526 1210491951 34118 SRX24912665 SRS21618599 SRA1898111 Harvard University Harvard University                       B   usable mapping rate illumina nextseq unknown other trueseq sc single_cell_droplet indrops   United States 2024-06-13 Multi-stage Embryo Whole Organism All anatomical structures

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CREATE TABLE run_metadata("run.accession" VARCHAR, "experiment.accession" VARCHAR, "sample.accession" VARCHAR, "study.accession" VARCHAR, bioproject VARCHAR, "study.title" VARCHAR, "study.alias" VARCHAR, "study.type" VARCHAR, "study.abstract" VARCHAR, "study.attributes" VARCHAR, "study.PMIDs" VARCHAR, "sample.description" VARCHAR, "sample.title" VARCHAR, "sample.alias" VARCHAR, "sample.centername" VARCHAR, "sample.attributes" VARCHAR, "GEOsample.title" VARCHAR, "GEOsample.dataprocessing" VARCHAR, "GEOsample.source" VARCHAR, "GEOsample.treatmentprotocol" VARCHAR, "GEOsample.extractprotocol" VARCHAR, "GEOsample.growthprotocol" VARCHAR, "GEOsample.characteristics" VARCHAR, "GEOsample.accession" VARCHAR, "experiment.title" VARCHAR, "experiment.alias" VARCHAR, "experiment.library_name" VARCHAR, "experiment.design_description" VARCHAR, "experiment.library_construction_protocol" VARCHAR, "experiment.attributes" VARCHAR, "experiment.library_strategy" VARCHAR, "experiment.library_source" VARCHAR, "experiment.library_selection" VARCHAR, "experiment.library_layout" VARCHAR, "experiment.platform" VARCHAR, "experiment.instrument_model" VARCHAR, "experiment.spot_descriptor" VARCHAR, "experiment.study_ref" VARCHAR, "run.title" VARCHAR, "run.attributes" VARCHAR, "run.filename" VARCHAR, "run.semantic_name" VARCHAR, "run.total_bases" DOUBLE, "run.total_spots" DOUBLE, "run.alias" VARCHAR, "run.read_lengths" VARCHAR, "run.base_counts" VARCHAR, "run.r1_length" BIGINT, "run.r2_length" BIGINT, "run.r3_length" BIGINT, "run.r4_length" BIGINT, "run.Acount" BIGINT, "run.Ccount" BIGINT, "run.Gcount" BIGINT, "run.Tcount" BIGINT, "run.Ncount" BIGINT, "run.experiment" VARCHAR, "run.pool_member" VARCHAR, "submission.accession" VARCHAR, "submission.srasource" VARCHAR, "submission.bioprojectsource" VARCHAR, "seqdetective.n_mates" BIGINT, "seqdetective.mapping_rate.mate1" DOUBLE, "seqdetective.mapping_rate.mate2" DOUBLE, "seqdetective.nofeature_rate.mate1" DOUBLE, "seqdetective.nofeature_rate.mate2" DOUBLE, "seqdetective.sparsity.mate1" DOUBLE, "seqdetective.sparsity.mate2" DOUBLE, "seqdetective.pos_strand_rate.mate1" DOUBLE, "seqdetective.pos_strand_rate.mate2" DOUBLE, "seqdetective.readlen.mate1" BIGINT, "seqdetective.readlen.mate2" BIGINT, "seqdetective.judgement.mate1" VARCHAR, "seqdetective.judgement.mate2" VARCHAR, "seqdetective.judgement.reason" VARCHAR, platform_family VARCHAR, instrument_generation VARCHAR, read_bias VARCHAR, selection_class VARCHAR, prep_kit VARCHAR, sc_or_bulk VARCHAR, tech_class VARCHAR, technology VARCHAR, tech_variant VARCHAR, "submission.bioprojectsource.country" VARCHAR, earliest_date DATE, devstage_curation VARCHAR, devstage_curation_coarse VARCHAR, tissue_curation VARCHAR, tissue_curation_coarse VARCHAR);;
CREATE INDEX idx_run_bioproject ON run_metadata(bioproject);;
CREATE INDEX idx_run_run_accession ON run_metadata("run.accession");;