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
60662,SRR12474621,SRX8968761,SRS7224498,SRP278034,PRJNA657343,Satb2 acts as a gatekeeper for gene regulatory transitions during early embryonic development,PRJNA657343,Other,Comprehensive integration of transcriptome  genome wide occupancy and chromatin accessibility profiles in satb2 loss of function and gain of function systems to discover novel and evolutionary conserved molecular interplays between Satb2 and the genetic drivers of neurogenesis and neural crest development program.,,,,scRNAseq FLAG SATB2,scRNAseq FLAG SATB2 dome,,strain:TU|isolate:Satb2 OE|breed:not applicable|cultivar:not applicable|ecotype:not applicable|age:4.5 hpf|dev stage:Dome|sex:not applicable|tissue:whole embryo|Replicate:replicate=FLAG SATB2 dome|BioSampleModel:Model organism or animal,,,,,,,,,scRNAseq FLAG SATB2,scRNAseq FLAG SATB2 dome,scRNAseq FLAG SATB2 dome,10x chromium 3 mRNA,,,OTHER,TRANSCRIPTOMIC SINGLE CELL,cDNA,PAIRED,ILLUMINA,NextSeq 550,,SRP278034,,loader:fastq load.py|options:  platform=Illumina   readTypes=TTB   read1PairFiles=scRNAseq FLAG SATB2 dome I1 sorted.fastq   read2PairFiles=scRNAseq FLAG SATB2 dome R1 sorted.fastq   read3PairFiles=scRNAseq FLAG SATB2 dome R2 sorted.fastq,scRNAseq_FLAG_SATB2_dome_I1_sorted.fastq scRNAseq_FLAG_SATB2_dome_R1_sorted.fastq scRNAseq_FLAG_SATB2_dome_R2_sorted.fastq,fastq fastq fastq,47518418346.0,340198768.0,scRNAseq FLAG SATB2 dome I1.fastq.gz,0:8 1:30.68 2:101,A:13016292601;C:10075463201;G:10532642161;T:13868229463;N:25790920,8,30,101,,13016292601,10075463201,10532642161,13868229463,25790920,SRX8968761,SRS7224498,SRA1114017,IISER-PUNE|biology,IISER-PUNE,1,0.92339,,0.12479,,0.81485,,0.52987,,101,,B,,usable mapping rate,illumina,nextseq,unknown,cdna_unspecified,unknown,sc,single_cell_droplet,10x,,India,2020-09-01,Blastula,Embryo,Whole Organism,All anatomical structures
62848,SRR13402902,SRX9821929,SRS8004722,SRP301260,PRJNA691237,Smad4 controls signaling robustness and morphogenesis by differentially contributing to the Nodal and BMP pathways,GSE164574,Other,The goal of this scRNA seq study was to characterize the pseudo spatial distribution of Nodal and BMP effectors SMADs expression prior to the onset of gastrulation Overall design: scRNA seq was performed on Zebrafish embryos at sphere stage,,pubmed:34737283,,Zsphere scRNA,GSM5014336,,tissue:Zebrafish embryos|Stage:sphere,Zsphere scRNA,We used cell ranger version 2.1.1 to deconvolve  align and quantify the per cell transcriptomes. Reads were aligned to version GRCz10 of the zebrafish genome with annotation release 89 from Ensembl software available from 10x genomics. From these alignments we were able to identify 2 644 high quality cells with a mean reads per cell value of 59 754 and a median genes per cell count of 3 807.  All other QC metrics were within thresholds set by 10x Genomics Genome build: GRCz10 release 89 from Ensembl Supplementary files format and content: tar file containing 10x counts matrix  features and barcode data,Zebrafish embryos,,At high stage  embryos were transferred in agar coated 6 cm petri dish and manually dechorionated in embryo media. At sphere stage  the embryo media was sequentially replaced with Dulbecco Modified Eagle’s Medium DMEM F 12 with 0.04% BSA and the blastula cups were dissected away from 25 embryos and transferred to an Eppendorf tube containing 100µl of DMEM F12 +0.04% BSA. The cell suspension was pelleted at 400 rcf for 30sec. The media was then removed and cells were suspended in 200µl of ice cold PBS+0.04% BSA to which 800µl of MeOH were added dropwise. Samples were kept in ice for 15 minutes and finally transferred to  20°C until use. For rehydration samples were kept in ice for 15 min and cells were pelleted at 1000rcf for 5 min at 4°C. Cells were resuspended in 100µl ice cold PBS + 0.04% BSA and the cell suspension was loaded into 10X Genomics Single Cell 3′ Chip The quality and concentration of the cell suspension was measured using an automatic cell counter. For sequencing  the ample was diluted to a concentration of 1000 cells/ul and approximately 10 000 cells were loaded into a Chromium Chip for use in the 10X Chromium Controller. The library was sequenced on a HiSeq 4000. 10x Chromium Single Cell three prime,Zebrafish embryos were grown in embryo media  for dissection of the blastula cup  embryos were transferred in DMEM F12 media,Stage:sphere,GSM5014336,GSM5014336: Zsphere scRNA; Danio rerio; RNA Seq,GSM5014336,,1,At high stage  embryos were transferred in agar coated 6 cm petri dish and manually dechorionated in embryo media. At sphere stage  the embryo media was sequentially replaced with Dulbecco Modified Eagle's Medium DMEM F 12 with 0.04% BSA and the blastula cups were dissected away from 25 embryos and transferred to an Eppendorf tube containing 100µl of DMEM F12 +0.04% BSA. The cell suspension was pelleted at 400 rcf for 30sec. The media was then removed and cells were suspended in 200µl of ice cold PBS+0.04% BSA to which 800µl of MeOH were added dropwise. Samples were kept in ice for 15 minutes and finally transferred to  20°C until use. For rehydration samples were kept in ice for 15 min and cells were pelleted at 1000rcf for 5 min at 4°C. Cells were resuspended in 100µl ice cold PBS + 0.04% BSA and the cell suspension was loaded into 10X Genomics Single Cell 3′ Chip The quality and concentration of the cell suspension was measured using an automatic cell counter. For sequencing  the ample was diluted to a concentration of 1000 cells/ul and approximately 10 000 cells were loaded into a Chromium Chip for use in the 10X Chromium Controller. The library was sequenced on a HiSeq 4000. 10x Chromium Single Cell three prime,GEO Accession:GSM5014336,RNA-Seq,TRANSCRIPTOMIC,cDNA,PAIRED,ILLUMINA,Illumina HiSeq 4000,,SRP301260,,loader:fastq load.py|options:  platform=Illumina   readTypes=TTB   read1PairFiles=GUG739A4 S2 L001 I1 001.fastq.gz   read2PairFiles=GUG739A4 S2 L001 R1 001.fastq.gz   read3PairFiles=GUG739A4 S2 L001 R2 001.fastq.gz,GUG739A4_S2_L001_I1_001.fastq.gz GUG739A4_S2_L001_R1_001.fastq.gz GUG739A4_S2_L001_R2_001.fastq.gz,fastq fastq fastq,5235400170.0,24930477.0,GSM5014336 r1,0:8 1:101 2:101,A:1177347684;C:925887308;G:939712357;T:2191666659;N:786162,8,101,101,,1177347684,925887308,939712357,2191666659,786162,SRX9821929,SRS8004722,SRA1182478,GEO,"Bioinformatics, Bioinformatics, The Francis Crick Institute",1,0.91081,,0.11647,,0.78459,,0.5629,,101,,B,,usable mapping rate,illumina,hiseq_era,unknown,cdna_unspecified,unknown,sc,single_cell_droplet,10x,,United Kingdom,2021-01-11,Blastula,Embryo,Embryo Imprecise,All anatomical structures
62849,SRR13402903,SRX9821929,SRS8004722,SRP301260,PRJNA691237,Smad4 controls signaling robustness and morphogenesis by differentially contributing to the Nodal and BMP pathways,GSE164574,Other,The goal of this scRNA seq study was to characterize the pseudo spatial distribution of Nodal and BMP effectors SMADs expression prior to the onset of gastrulation Overall design: scRNA seq was performed on Zebrafish embryos at sphere stage,,pubmed:34737283,,Zsphere scRNA,GSM5014336,,tissue:Zebrafish embryos|Stage:sphere,Zsphere scRNA,We used cell ranger version 2.1.1 to deconvolve  align and quantify the per cell transcriptomes. Reads were aligned to version GRCz10 of the zebrafish genome with annotation release 89 from Ensembl software available from 10x genomics. From these alignments we were able to identify 2 644 high quality cells with a mean reads per cell value of 59 754 and a median genes per cell count of 3 807.  All other QC metrics were within thresholds set by 10x Genomics Genome build: GRCz10 release 89 from Ensembl Supplementary files format and content: tar file containing 10x counts matrix  features and barcode data,Zebrafish embryos,,At high stage  embryos were transferred in agar coated 6 cm petri dish and manually dechorionated in embryo media. At sphere stage  the embryo media was sequentially replaced with Dulbecco Modified Eagle’s Medium DMEM F 12 with 0.04% BSA and the blastula cups were dissected away from 25 embryos and transferred to an Eppendorf tube containing 100µl of DMEM F12 +0.04% BSA. The cell suspension was pelleted at 400 rcf for 30sec. The media was then removed and cells were suspended in 200µl of ice cold PBS+0.04% BSA to which 800µl of MeOH were added dropwise. Samples were kept in ice for 15 minutes and finally transferred to  20°C until use. For rehydration samples were kept in ice for 15 min and cells were pelleted at 1000rcf for 5 min at 4°C. Cells were resuspended in 100µl ice cold PBS + 0.04% BSA and the cell suspension was loaded into 10X Genomics Single Cell 3′ Chip The quality and concentration of the cell suspension was measured using an automatic cell counter. For sequencing  the ample was diluted to a concentration of 1000 cells/ul and approximately 10 000 cells were loaded into a Chromium Chip for use in the 10X Chromium Controller. The library was sequenced on a HiSeq 4000. 10x Chromium Single Cell three prime,Zebrafish embryos were grown in embryo media  for dissection of the blastula cup  embryos were transferred in DMEM F12 media,Stage:sphere,GSM5014336,GSM5014336: Zsphere scRNA; Danio rerio; RNA Seq,GSM5014336,,1,At high stage  embryos were transferred in agar coated 6 cm petri dish and manually dechorionated in embryo media. At sphere stage  the embryo media was sequentially replaced with Dulbecco Modified Eagle's Medium DMEM F 12 with 0.04% BSA and the blastula cups were dissected away from 25 embryos and transferred to an Eppendorf tube containing 100µl of DMEM F12 +0.04% BSA. The cell suspension was pelleted at 400 rcf for 30sec. The media was then removed and cells were suspended in 200µl of ice cold PBS+0.04% BSA to which 800µl of MeOH were added dropwise. Samples were kept in ice for 15 minutes and finally transferred to  20°C until use. For rehydration samples were kept in ice for 15 min and cells were pelleted at 1000rcf for 5 min at 4°C. Cells were resuspended in 100µl ice cold PBS + 0.04% BSA and the cell suspension was loaded into 10X Genomics Single Cell 3′ Chip The quality and concentration of the cell suspension was measured using an automatic cell counter. For sequencing  the ample was diluted to a concentration of 1000 cells/ul and approximately 10 000 cells were loaded into a Chromium Chip for use in the 10X Chromium Controller. The library was sequenced on a HiSeq 4000. 10x Chromium Single Cell three prime,GEO Accession:GSM5014336,RNA-Seq,TRANSCRIPTOMIC,cDNA,PAIRED,ILLUMINA,Illumina HiSeq 4000,,SRP301260,,loader:fastq load.py|options:  platform=Illumina   readTypes=TTB   read1PairFiles=GUG739A4 S2 L002 I1 001.fastq.gz   read2PairFiles=GUG739A4 S2 L002 R1 001.fastq.gz   read3PairFiles=GUG739A4 S2 L002 R2 001.fastq.gz,GUG739A4_S2_L002_I1_001.fastq.gz GUG739A4_S2_L002_R1_001.fastq.gz GUG739A4_S2_L002_R2_001.fastq.gz,fastq fastq fastq,5543517210.0,26397701.0,GSM5014336 r2,0:8 1:101 2:101,A:1240643673;C:980694158;G:992958309;T:2328732820;N:488250,8,101,101,,1240643673,980694158,992958309,2328732820,488250,SRX9821929,SRS8004722,SRA1182478,GEO,"Bioinformatics, Bioinformatics, The Francis Crick Institute",1,0.91665,,0.11529,,0.78472,,0.58007,,101,,B,,usable mapping rate,illumina,hiseq_era,unknown,cdna_unspecified,unknown,sc,single_cell_droplet,10x,,United Kingdom,2021-01-11,Blastula,Embryo,Embryo Imprecise,All anatomical structures
62850,SRR13402904,SRX9821929,SRS8004722,SRP301260,PRJNA691237,Smad4 controls signaling robustness and morphogenesis by differentially contributing to the Nodal and BMP pathways,GSE164574,Other,The goal of this scRNA seq study was to characterize the pseudo spatial distribution of Nodal and BMP effectors SMADs expression prior to the onset of gastrulation Overall design: scRNA seq was performed on Zebrafish embryos at sphere stage,,pubmed:34737283,,Zsphere scRNA,GSM5014336,,tissue:Zebrafish embryos|Stage:sphere,Zsphere scRNA,We used cell ranger version 2.1.1 to deconvolve  align and quantify the per cell transcriptomes. Reads were aligned to version GRCz10 of the zebrafish genome with annotation release 89 from Ensembl software available from 10x genomics. From these alignments we were able to identify 2 644 high quality cells with a mean reads per cell value of 59 754 and a median genes per cell count of 3 807.  All other QC metrics were within thresholds set by 10x Genomics Genome build: GRCz10 release 89 from Ensembl Supplementary files format and content: tar file containing 10x counts matrix  features and barcode data,Zebrafish embryos,,At high stage  embryos were transferred in agar coated 6 cm petri dish and manually dechorionated in embryo media. At sphere stage  the embryo media was sequentially replaced with Dulbecco Modified Eagle’s Medium DMEM F 12 with 0.04% BSA and the blastula cups were dissected away from 25 embryos and transferred to an Eppendorf tube containing 100µl of DMEM F12 +0.04% BSA. The cell suspension was pelleted at 400 rcf for 30sec. The media was then removed and cells were suspended in 200µl of ice cold PBS+0.04% BSA to which 800µl of MeOH were added dropwise. Samples were kept in ice for 15 minutes and finally transferred to  20°C until use. For rehydration samples were kept in ice for 15 min and cells were pelleted at 1000rcf for 5 min at 4°C. Cells were resuspended in 100µl ice cold PBS + 0.04% BSA and the cell suspension was loaded into 10X Genomics Single Cell 3′ Chip The quality and concentration of the cell suspension was measured using an automatic cell counter. For sequencing  the ample was diluted to a concentration of 1000 cells/ul and approximately 10 000 cells were loaded into a Chromium Chip for use in the 10X Chromium Controller. The library was sequenced on a HiSeq 4000. 10x Chromium Single Cell three prime,Zebrafish embryos were grown in embryo media  for dissection of the blastula cup  embryos were transferred in DMEM F12 media,Stage:sphere,GSM5014336,GSM5014336: Zsphere scRNA; Danio rerio; RNA Seq,GSM5014336,,1,At high stage  embryos were transferred in agar coated 6 cm petri dish and manually dechorionated in embryo media. At sphere stage  the embryo media was sequentially replaced with Dulbecco Modified Eagle's Medium DMEM F 12 with 0.04% BSA and the blastula cups were dissected away from 25 embryos and transferred to an Eppendorf tube containing 100µl of DMEM F12 +0.04% BSA. The cell suspension was pelleted at 400 rcf for 30sec. The media was then removed and cells were suspended in 200µl of ice cold PBS+0.04% BSA to which 800µl of MeOH were added dropwise. Samples were kept in ice for 15 minutes and finally transferred to  20°C until use. For rehydration samples were kept in ice for 15 min and cells were pelleted at 1000rcf for 5 min at 4°C. Cells were resuspended in 100µl ice cold PBS + 0.04% BSA and the cell suspension was loaded into 10X Genomics Single Cell 3′ Chip The quality and concentration of the cell suspension was measured using an automatic cell counter. For sequencing  the ample was diluted to a concentration of 1000 cells/ul and approximately 10 000 cells were loaded into a Chromium Chip for use in the 10X Chromium Controller. The library was sequenced on a HiSeq 4000. 10x Chromium Single Cell three prime,GEO Accession:GSM5014336,RNA-Seq,TRANSCRIPTOMIC,cDNA,PAIRED,ILLUMINA,Illumina HiSeq 4000,,SRP301260,,loader:fastq load.py|options:  platform=Illumina   readTypes=TTB   read1PairFiles=GUG739A4 S2 L003 I1 001.fastq.gz   read2PairFiles=GUG739A4 S2 L003 R1 001.fastq.gz   read3PairFiles=GUG739A4 S2 L003 R2 001.fastq.gz,GUG739A4_S2_L003_I1_001.fastq.gz GUG739A4_S2_L003_R1_001.fastq.gz GUG739A4_S2_L003_R2_001.fastq.gz,fastq fastq fastq,5572550130.0,26535953.0,GSM5014336 r3,0:8 1:101 2:101,A:1249457035;C:994601699;G:1005584984;T:2322470542;N:435870,8,101,101,,1249457035,994601699,1005584984,2322470542,435870,SRX9821929,SRS8004722,SRA1182478,GEO,"Bioinformatics, Bioinformatics, The Francis Crick Institute",1,0.90814,,0.11693,,0.78565,,0.57928,,101,,B,,usable mapping rate,illumina,hiseq_era,unknown,cdna_unspecified,unknown,sc,single_cell_droplet,10x,,United Kingdom,2021-01-11,Blastula,Embryo,Embryo Imprecise,All anatomical structures
62851,SRR13402905,SRX9821929,SRS8004722,SRP301260,PRJNA691237,Smad4 controls signaling robustness and morphogenesis by differentially contributing to the Nodal and BMP pathways,GSE164574,Other,The goal of this scRNA seq study was to characterize the pseudo spatial distribution of Nodal and BMP effectors SMADs expression prior to the onset of gastrulation Overall design: scRNA seq was performed on Zebrafish embryos at sphere stage,,pubmed:34737283,,Zsphere scRNA,GSM5014336,,tissue:Zebrafish embryos|Stage:sphere,Zsphere scRNA,We used cell ranger version 2.1.1 to deconvolve  align and quantify the per cell transcriptomes. Reads were aligned to version GRCz10 of the zebrafish genome with annotation release 89 from Ensembl software available from 10x genomics. From these alignments we were able to identify 2 644 high quality cells with a mean reads per cell value of 59 754 and a median genes per cell count of 3 807.  All other QC metrics were within thresholds set by 10x Genomics Genome build: GRCz10 release 89 from Ensembl Supplementary files format and content: tar file containing 10x counts matrix  features and barcode data,Zebrafish embryos,,At high stage  embryos were transferred in agar coated 6 cm petri dish and manually dechorionated in embryo media. At sphere stage  the embryo media was sequentially replaced with Dulbecco Modified Eagle’s Medium DMEM F 12 with 0.04% BSA and the blastula cups were dissected away from 25 embryos and transferred to an Eppendorf tube containing 100µl of DMEM F12 +0.04% BSA. The cell suspension was pelleted at 400 rcf for 30sec. The media was then removed and cells were suspended in 200µl of ice cold PBS+0.04% BSA to which 800µl of MeOH were added dropwise. Samples were kept in ice for 15 minutes and finally transferred to  20°C until use. For rehydration samples were kept in ice for 15 min and cells were pelleted at 1000rcf for 5 min at 4°C. Cells were resuspended in 100µl ice cold PBS + 0.04% BSA and the cell suspension was loaded into 10X Genomics Single Cell 3′ Chip The quality and concentration of the cell suspension was measured using an automatic cell counter. For sequencing  the ample was diluted to a concentration of 1000 cells/ul and approximately 10 000 cells were loaded into a Chromium Chip for use in the 10X Chromium Controller. The library was sequenced on a HiSeq 4000. 10x Chromium Single Cell three prime,Zebrafish embryos were grown in embryo media  for dissection of the blastula cup  embryos were transferred in DMEM F12 media,Stage:sphere,GSM5014336,GSM5014336: Zsphere scRNA; Danio rerio; RNA Seq,GSM5014336,,1,At high stage  embryos were transferred in agar coated 6 cm petri dish and manually dechorionated in embryo media. At sphere stage  the embryo media was sequentially replaced with Dulbecco Modified Eagle's Medium DMEM F 12 with 0.04% BSA and the blastula cups were dissected away from 25 embryos and transferred to an Eppendorf tube containing 100µl of DMEM F12 +0.04% BSA. The cell suspension was pelleted at 400 rcf for 30sec. The media was then removed and cells were suspended in 200µl of ice cold PBS+0.04% BSA to which 800µl of MeOH were added dropwise. Samples were kept in ice for 15 minutes and finally transferred to  20°C until use. For rehydration samples were kept in ice for 15 min and cells were pelleted at 1000rcf for 5 min at 4°C. Cells were resuspended in 100µl ice cold PBS + 0.04% BSA and the cell suspension was loaded into 10X Genomics Single Cell 3′ Chip The quality and concentration of the cell suspension was measured using an automatic cell counter. For sequencing  the ample was diluted to a concentration of 1000 cells/ul and approximately 10 000 cells were loaded into a Chromium Chip for use in the 10X Chromium Controller. The library was sequenced on a HiSeq 4000. 10x Chromium Single Cell three prime,GEO Accession:GSM5014336,RNA-Seq,TRANSCRIPTOMIC,cDNA,PAIRED,ILLUMINA,Illumina HiSeq 4000,,SRP301260,,loader:fastq load.py|options:  platform=Illumina   readTypes=TTB   read1PairFiles=GUG739A4 S2 L004 I1 001.fastq.gz   read2PairFiles=GUG739A4 S2 L004 R1 001.fastq.gz   read3PairFiles=GUG739A4 S2 L004 R2 001.fastq.gz,GUG739A4_S2_L004_I1_001.fastq.gz GUG739A4_S2_L004_R1_001.fastq.gz GUG739A4_S2_L004_R2_001.fastq.gz,fastq fastq fastq,5614329210.0,26734901.0,GSM5014336 r4,0:8 1:101 2:101,A:1257192667;C:993041786;G:1007516062;T:2349738132;N:6840563,8,101,101,,1257192667,993041786,1007516062,2349738132,6840563,SRX9821929,SRS8004722,SRA1182478,GEO,"Bioinformatics, Bioinformatics, The Francis Crick Institute",1,0.91345,,0.11553,,0.78632,,0.58232,,101,,B,,usable mapping rate,illumina,hiseq_era,unknown,cdna_unspecified,unknown,sc,single_cell_droplet,10x,,United Kingdom,2021-01-11,Blastula,Embryo,Embryo Imprecise,All anatomical structures
62852,SRR13402906,SRX9821929,SRS8004722,SRP301260,PRJNA691237,Smad4 controls signaling robustness and morphogenesis by differentially contributing to the Nodal and BMP pathways,GSE164574,Other,The goal of this scRNA seq study was to characterize the pseudo spatial distribution of Nodal and BMP effectors SMADs expression prior to the onset of gastrulation Overall design: scRNA seq was performed on Zebrafish embryos at sphere stage,,pubmed:34737283,,Zsphere scRNA,GSM5014336,,tissue:Zebrafish embryos|Stage:sphere,Zsphere scRNA,We used cell ranger version 2.1.1 to deconvolve  align and quantify the per cell transcriptomes. Reads were aligned to version GRCz10 of the zebrafish genome with annotation release 89 from Ensembl software available from 10x genomics. From these alignments we were able to identify 2 644 high quality cells with a mean reads per cell value of 59 754 and a median genes per cell count of 3 807.  All other QC metrics were within thresholds set by 10x Genomics Genome build: GRCz10 release 89 from Ensembl Supplementary files format and content: tar file containing 10x counts matrix  features and barcode data,Zebrafish embryos,,At high stage  embryos were transferred in agar coated 6 cm petri dish and manually dechorionated in embryo media. At sphere stage  the embryo media was sequentially replaced with Dulbecco Modified Eagle’s Medium DMEM F 12 with 0.04% BSA and the blastula cups were dissected away from 25 embryos and transferred to an Eppendorf tube containing 100µl of DMEM F12 +0.04% BSA. The cell suspension was pelleted at 400 rcf for 30sec. The media was then removed and cells were suspended in 200µl of ice cold PBS+0.04% BSA to which 800µl of MeOH were added dropwise. Samples were kept in ice for 15 minutes and finally transferred to  20°C until use. For rehydration samples were kept in ice for 15 min and cells were pelleted at 1000rcf for 5 min at 4°C. Cells were resuspended in 100µl ice cold PBS + 0.04% BSA and the cell suspension was loaded into 10X Genomics Single Cell 3′ Chip The quality and concentration of the cell suspension was measured using an automatic cell counter. For sequencing  the ample was diluted to a concentration of 1000 cells/ul and approximately 10 000 cells were loaded into a Chromium Chip for use in the 10X Chromium Controller. The library was sequenced on a HiSeq 4000. 10x Chromium Single Cell three prime,Zebrafish embryos were grown in embryo media  for dissection of the blastula cup  embryos were transferred in DMEM F12 media,Stage:sphere,GSM5014336,GSM5014336: Zsphere scRNA; Danio rerio; RNA Seq,GSM5014336,,1,At high stage  embryos were transferred in agar coated 6 cm petri dish and manually dechorionated in embryo media. At sphere stage  the embryo media was sequentially replaced with Dulbecco Modified Eagle's Medium DMEM F 12 with 0.04% BSA and the blastula cups were dissected away from 25 embryos and transferred to an Eppendorf tube containing 100µl of DMEM F12 +0.04% BSA. The cell suspension was pelleted at 400 rcf for 30sec. The media was then removed and cells were suspended in 200µl of ice cold PBS+0.04% BSA to which 800µl of MeOH were added dropwise. Samples were kept in ice for 15 minutes and finally transferred to  20°C until use. For rehydration samples were kept in ice for 15 min and cells were pelleted at 1000rcf for 5 min at 4°C. Cells were resuspended in 100µl ice cold PBS + 0.04% BSA and the cell suspension was loaded into 10X Genomics Single Cell 3′ Chip The quality and concentration of the cell suspension was measured using an automatic cell counter. For sequencing  the ample was diluted to a concentration of 1000 cells/ul and approximately 10 000 cells were loaded into a Chromium Chip for use in the 10X Chromium Controller. The library was sequenced on a HiSeq 4000. 10x Chromium Single Cell three prime,GEO Accession:GSM5014336,RNA-Seq,TRANSCRIPTOMIC,cDNA,PAIRED,ILLUMINA,Illumina HiSeq 4000,,SRP301260,,loader:fastq load.py|options:  platform=Illumina   readTypes=TTB   read1PairFiles=GUG739A4 S2 L005 I1 001.fastq.gz   read2PairFiles=GUG739A4 S2 L005 R1 001.fastq.gz   read3PairFiles=GUG739A4 S2 L005 R2 001.fastq.gz,GUG739A4_S2_L005_I1_001.fastq.gz GUG739A4_S2_L005_R1_001.fastq.gz GUG739A4_S2_L005_R2_001.fastq.gz,fastq fastq fastq,5660581290.0,26955149.0,GSM5014336 r5,0:8 1:101 2:101,A:1268509148;C:1023948185;G:1033027319;T:2334888489;N:208149,8,101,101,,1268509148,1023948185,1033027319,2334888489,208149,SRX9821929,SRS8004722,SRA1182478,GEO,"Bioinformatics, Bioinformatics, The Francis Crick Institute",1,0.91009,,0.11843,,0.78599,,0.57826,,101,,B,,usable mapping rate,illumina,hiseq_era,unknown,cdna_unspecified,unknown,sc,single_cell_droplet,10x,,United Kingdom,2021-01-11,Blastula,Embryo,Embryo Imprecise,All anatomical structures
62853,SRR13402907,SRX9821929,SRS8004722,SRP301260,PRJNA691237,Smad4 controls signaling robustness and morphogenesis by differentially contributing to the Nodal and BMP pathways,GSE164574,Other,The goal of this scRNA seq study was to characterize the pseudo spatial distribution of Nodal and BMP effectors SMADs expression prior to the onset of gastrulation Overall design: scRNA seq was performed on Zebrafish embryos at sphere stage,,pubmed:34737283,,Zsphere scRNA,GSM5014336,,tissue:Zebrafish embryos|Stage:sphere,Zsphere scRNA,We used cell ranger version 2.1.1 to deconvolve  align and quantify the per cell transcriptomes. Reads were aligned to version GRCz10 of the zebrafish genome with annotation release 89 from Ensembl software available from 10x genomics. From these alignments we were able to identify 2 644 high quality cells with a mean reads per cell value of 59 754 and a median genes per cell count of 3 807.  All other QC metrics were within thresholds set by 10x Genomics Genome build: GRCz10 release 89 from Ensembl Supplementary files format and content: tar file containing 10x counts matrix  features and barcode data,Zebrafish embryos,,At high stage  embryos were transferred in agar coated 6 cm petri dish and manually dechorionated in embryo media. At sphere stage  the embryo media was sequentially replaced with Dulbecco Modified Eagle’s Medium DMEM F 12 with 0.04% BSA and the blastula cups were dissected away from 25 embryos and transferred to an Eppendorf tube containing 100µl of DMEM F12 +0.04% BSA. The cell suspension was pelleted at 400 rcf for 30sec. The media was then removed and cells were suspended in 200µl of ice cold PBS+0.04% BSA to which 800µl of MeOH were added dropwise. Samples were kept in ice for 15 minutes and finally transferred to  20°C until use. For rehydration samples were kept in ice for 15 min and cells were pelleted at 1000rcf for 5 min at 4°C. Cells were resuspended in 100µl ice cold PBS + 0.04% BSA and the cell suspension was loaded into 10X Genomics Single Cell 3′ Chip The quality and concentration of the cell suspension was measured using an automatic cell counter. For sequencing  the ample was diluted to a concentration of 1000 cells/ul and approximately 10 000 cells were loaded into a Chromium Chip for use in the 10X Chromium Controller. The library was sequenced on a HiSeq 4000. 10x Chromium Single Cell three prime,Zebrafish embryos were grown in embryo media  for dissection of the blastula cup  embryos were transferred in DMEM F12 media,Stage:sphere,GSM5014336,GSM5014336: Zsphere scRNA; Danio rerio; RNA Seq,GSM5014336,,1,At high stage  embryos were transferred in agar coated 6 cm petri dish and manually dechorionated in embryo media. At sphere stage  the embryo media was sequentially replaced with Dulbecco Modified Eagle's Medium DMEM F 12 with 0.04% BSA and the blastula cups were dissected away from 25 embryos and transferred to an Eppendorf tube containing 100µl of DMEM F12 +0.04% BSA. The cell suspension was pelleted at 400 rcf for 30sec. The media was then removed and cells were suspended in 200µl of ice cold PBS+0.04% BSA to which 800µl of MeOH were added dropwise. Samples were kept in ice for 15 minutes and finally transferred to  20°C until use. For rehydration samples were kept in ice for 15 min and cells were pelleted at 1000rcf for 5 min at 4°C. Cells were resuspended in 100µl ice cold PBS + 0.04% BSA and the cell suspension was loaded into 10X Genomics Single Cell 3′ Chip The quality and concentration of the cell suspension was measured using an automatic cell counter. For sequencing  the ample was diluted to a concentration of 1000 cells/ul and approximately 10 000 cells were loaded into a Chromium Chip for use in the 10X Chromium Controller. The library was sequenced on a HiSeq 4000. 10x Chromium Single Cell three prime,GEO Accession:GSM5014336,RNA-Seq,TRANSCRIPTOMIC,cDNA,PAIRED,ILLUMINA,Illumina HiSeq 4000,,SRP301260,,loader:fastq load.py|options:  platform=Illumina   readTypes=TTB   read1PairFiles=GUG739A4 S2 L006 I1 001.fastq.gz   read2PairFiles=GUG739A4 S2 L006 R1 001.fastq.gz   read3PairFiles=GUG739A4 S2 L006 R2 001.fastq.gz,GUG739A4_S2_L006_I1_001.fastq.gz GUG739A4_S2_L006_R1_001.fastq.gz GUG739A4_S2_L006_R2_001.fastq.gz,fastq fastq fastq,5551742700.0,26436870.0,GSM5014336 r6,0:8 1:101 2:101,A:1244015771;C:994654836;G:1005415645;T:2307255998;N:400450,8,101,101,,1244015771,994654836,1005415645,2307255998,400450,SRX9821929,SRS8004722,SRA1182478,GEO,"Bioinformatics, Bioinformatics, The Francis Crick Institute",1,0.90906,,0.11635,,0.78421,,0.5674,,101,,B,,usable mapping rate,illumina,hiseq_era,unknown,cdna_unspecified,unknown,sc,single_cell_droplet,10x,,United Kingdom,2021-01-11,Blastula,Embryo,Embryo Imprecise,All anatomical structures
63042,SRR13565184,SRX9963428,SRS8137095,SRP303506,PRJNA695274,Single cell response landscape of graded Nodal signaling  in zebrafish explants [single cell RNA seq],GSE165653,Transcriptome Analysis,Purpose: To construct cell landscape of graded Nodal signaling  in zebrafish explants. Methods:  Nodal injected explants injected with 10pg ndr2 mRNA were harvested at xxxhpf  5hpf  6hpf  8hpf  10hpf  24hpf. Libraries were prepared using Chromium Controller and Chromium Single Cell three primeLibrary & Gel Bead Kit v2 10x Genomics  PN 120237 according to the manufacturer's protocol for 10000 cells recovery. Results: A total of 42 317 single cell transcriptomes were collected post stringent quality control measures. Conclusions: 19 Cell types from 6 developmental stages were identified  Nodal explant mainly contains the axial mesoderm prechordal plate and notochord and anterior posterior patterned head structures. Overall design: zebrafish explants injected with 10pg ndr2 mRNA were harvested at xxxhpf  5hpf  6hpf  8hpf  10hpf  24hpf for scRNA seq.,parent bioproject:PRJNA695267,pubmed:37729057,,zebrafish Nodal explants 5hpf,GSM5047612,,source name:zebrafish cells|strain:AB|tissue:embryonic cells|age:5hpf|treatment:injected with 10pg ndr2 mRNA,zebrafish Nodal explants 5hpf,Illumina sequencing reads were aligned to the zebrafish mRNA reference genome GRCz11 or GRCz10 using the 10x Genomics CellRanger pipeline version 2.1.1 with default parameters. Genome build: GRCz11/GRCz10 Supplementary files format and content: tar compressed  files included filtered gene bc matrices post running CellRanger pipeline,zebrafish cells,10pg of ndr2 mRNA was injected to one cell of embryonic animal pole at xxx cell stage. All embryos were incubated in 0.3x Danieau buffer until 1k stage  then  were transferred to Dulbecco's Modified Eagle Medium. Animal pole explants corresponding roughly to half of the blastula were incubated to 4hpf  5hpf  6hpf  8hpf  10hpf and 24hpf corresponding to embryonic developmental stage separately.,Libraries were prepared using Chromium Controller and Chromium Single Cell 3’Library & Gel Bead Kit v2 10x Genomics  PN 120237 according to the manufacturer’s protocol for 10000 cells recovery.,explants were cultured in a Petri dish coated with 1.5% agarose filled with Dulbecco's Modified Eagle Medium,strain:AB|tissue:embryonic cells|age:5hpf|treatment:injected with 10pg ndr2 mRNA,GSM5047612,GSM5047612: zebrafish Nodal explants 5hpf; Danio rerio; RNA Seq,GSM5047612,,1,Libraries were prepared using Chromium Controller and Chromium Single Cell three primeLibrary & Gel Bead Kit v2 10x Genomics  PN 120237 according to the manufacturer's protocol for 10000 cells recovery.,GEO Accession:GSM5047612,RNA-Seq,TRANSCRIPTOMIC,cDNA,PAIRED,ILLUMINA,Illumina NovaSeq 6000,,SRP303506,,,wt_cyc_5hpf_S1_L002_R1_001.fastq.gz wt_cyc_5hpf_S1_L002_R2_001.fastq.gz,fastq fastq,132074187896.0,437331748.0,GSM5047612 r1,0:151 1:151,A:33956635680;C:22833737374;G:24573359678;T:50703117357;N:7337807,151,151,,,33956635680,22833737374,24573359678,50703117357,7337807,SRX9963428,SRS8137095,SRA1188621,GEO,"Institute of genetics, Zhejiang University",2,0.25456,0.90744,0.02797,0.06895,0.96999,0.79732,0.57673,0.63217,151,151,T,B,mate1 technical by mapping diff,illumina,novaseq_era,unknown,cdna_unspecified,unknown,sc,single_cell_droplet,10x,,China,2021-01-27,Blastula,Embryo,Embryo Imprecise,All anatomical structures
63043,SRR13565183,SRX9963427,SRS8137094,SRP303506,PRJNA695274,Single cell response landscape of graded Nodal signaling  in zebrafish explants [single cell RNA seq],GSE165653,Transcriptome Analysis,Purpose: To construct cell landscape of graded Nodal signaling  in zebrafish explants. Methods:  Nodal injected explants injected with 10pg ndr2 mRNA were harvested at xxxhpf  5hpf  6hpf  8hpf  10hpf  24hpf. Libraries were prepared using Chromium Controller and Chromium Single Cell three primeLibrary & Gel Bead Kit v2 10x Genomics  PN 120237 according to the manufacturer's protocol for 10000 cells recovery. Results: A total of 42 317 single cell transcriptomes were collected post stringent quality control measures. Conclusions: 19 Cell types from 6 developmental stages were identified  Nodal explant mainly contains the axial mesoderm prechordal plate and notochord and anterior posterior patterned head structures. Overall design: zebrafish explants injected with 10pg ndr2 mRNA were harvested at xxxhpf  5hpf  6hpf  8hpf  10hpf  24hpf for scRNA seq.,parent bioproject:PRJNA695267,pubmed:37729057,,zebrafish Nodal explants 4hpf,GSM5047611,,source name:zebrafish cells|strain:AB|tissue:embryonic cells|age:4hpf|treatment:injected with 10pg ndr2 mRNA,zebrafish Nodal explants 4hpf,Illumina sequencing reads were aligned to the zebrafish mRNA reference genome GRCz11 or GRCz10 using the 10x Genomics CellRanger pipeline version 2.1.1 with default parameters. Genome build: GRCz11/GRCz10 Supplementary files format and content: tar compressed  files included filtered gene bc matrices post running CellRanger pipeline,zebrafish cells,10pg of ndr2 mRNA was injected to one cell of embryonic animal pole at xxx cell stage. All embryos were incubated in 0.3x Danieau buffer until 1k stage  then  were transferred to Dulbecco's Modified Eagle Medium. Animal pole explants corresponding roughly to half of the blastula were incubated to 4hpf  5hpf  6hpf  8hpf  10hpf and 24hpf corresponding to embryonic developmental stage separately.,Libraries were prepared using Chromium Controller and Chromium Single Cell 3’Library & Gel Bead Kit v2 10x Genomics  PN 120237 according to the manufacturer’s protocol for 10000 cells recovery.,explants were cultured in a Petri dish coated with 1.5% agarose filled with Dulbecco's Modified Eagle Medium,strain:AB|tissue:embryonic cells|age:4hpf|treatment:injected with 10pg ndr2 mRNA,GSM5047611,GSM5047611: zebrafish Nodal explants 4hpf; Danio rerio; RNA Seq,GSM5047611,,1,Libraries were prepared using Chromium Controller and Chromium Single Cell three primeLibrary & Gel Bead Kit v2 10x Genomics  PN 120237 according to the manufacturer's protocol for 10000 cells recovery.,GEO Accession:GSM5047611,RNA-Seq,TRANSCRIPTOMIC,cDNA,PAIRED,ILLUMINA,Illumina NovaSeq 6000,,SRP303506,,,wt_cyc_4hpf_S1_L007_R1_001.fastq.gz wt_cyc_4hpf_S1_L007_R2_001.fastq.gz,fastq fastq,131011503350.0,433812925.0,GSM5047611 r1,0:151 1:151,A:33733394074;C:20387214801;G:21857805230;T:55019780406;N:13308839,151,151,,,33733394074,20387214801,21857805230,55019780406,13308839,SRX9963427,SRS8137094,SRA1188621,GEO,"Institute of genetics, Zhejiang University",2,0.34825,0.89448,0.03615,0.09865,0.94456,0.77555,0.51973,0.48621,151,151,B,B,mate1-mate2 similar by mapping diff,illumina,novaseq_era,unknown,cdna_unspecified,unknown,sc,single_cell_droplet,10x,,China,2021-01-27,Blastula,Embryo,Embryo Imprecise,All anatomical structures
64431,SRR14703458,SRX11041470,SRS9110398,SRP322195,PRJNA734348,Methylome inheritance and enhancer dememorization construct an epigenetic gate safeguarding embryonic programs,GSE175951,Other,Unlike that of mammals  the total DNA methylome of many cold blooded vertebrates is globally inherited from gametes to early embryos. In zebrafish  this is however accompanied by sweeping “dememorization” of enhancers prior to fertilization for sperm and just post fertilization for oocyte  as they undergo full methylation and are not demethylated again until phylotypic stage. The significance of both global methylome inheritance and enhancer dememorization in early embryos remains largely unknown. Adding to the puzzles  the zygotic mutant zebrafish of dnmt1  the major DNA methylation maintenance methyltransferase  surprisingly can develop to term. To solve the role of DNA methylation in early development  we generated zebrafish embryos derived from dnmt1 knocking down oocytes using a recently developed method OMIS microinjection in situ  which successfully eliminated DNA methylation before zygotic genome activation. dnmt1 deficient embryos failed to initiate epiboly and died around gastrulation. This is in part caused by activation of immune response and p53 regulated apoptosis  likely triggered by the derepression of transposable elements. Single cell RNA seq further revealed defective differentiation in these mutants. DNA methylation is also required for the establishment of repressive histone marks H3K27me3 and H2AK119ub. Strikingly  the loss of DNA methylation leads to extensive derepression of somatic genes and enhancers  which acquire ectopic H3K27ac  accessible chromatin  and H3K4me3. These somatic enhancers are preferentially CG rich and are bound by CG containing TFs. By contrast  embryonic enhancers are generally CG poor  methylation insensitive  and are bound by CG less TFs. Hence  the global DNA methylome inheritance is essential for vertebrate early development  and enhancer dememorization resets an epigenetic gate that separates embryonic and somatic programs. Overall design: By employing MethylC seq  total RNA seq  scRNA seq  CUT&RUN  ChIP seq  and ATAC seq in control and dnmt1 mKD embryos at oocyte  256 cell  dome and shield stages in zebrafish  we interrogated the function of DNA methylome and its inheritance in early zebrafish development.,,pubmed:34936444,,Dome dnmt1 mKD scRNA seq rep2,GSM5351825,,source name:embryo|strain:AB|genotype:WT|developmental stage:Dome|tissue:embryo|treatment:microinjection with Rhodamine B and dnmt1 MO at oogenesis III stage GV,Dome dnmt1 mKD scRNA seq rep2,All STEM seq datasets were mapped to the danRer7 reference genome by Bismark Krueger and Andrews  2011. Reads were trimmed with cutadaptor Martin  2011 using parameters:   minimum length 20   pair filter=any. Alignments were performed with the following parameters:  N 1  X 600   score min L 0  0.6. Multi mapped reads and PCR duplicates were removed. bismark methylation extractor was used to calculate the DNA methylation level. Total RNA seq data were firstly processed using Trim Galore! with default parameters to trim the adapter containing and low quality reads. The filtered data were then mapped to the zebrafish reference genome danRer7 by STAR version: STAR 2.5.3a modified Dobin et al.  2013 with the following parameters:   outSAMstrandField intronMotif   outSAMattributes All   outSAMunmapped Within   outSAMattrIHstart 0   outWigStrand Stranded   outFilterMultimapNmax 20   twopassMode Basic. The gene expression level was normalized to fragments per kilobase of transcript per million mapped FPKM values using Cufflinks version 2.2.1 Trapnell et al.  2012. The scRNA seq data were processed with Cell Ranger 3.1 for genome alignment danRer10  transcript counting  and gene cell barcode matrix generation. Before the downstream analysis  cells with unique detected genes lower than <2 000 and higher than 6 000 genes were discarded  and cells with high percentage of mitochondrial genes >5% were removed too. Subtypes of cells were identified with Seurat 3.0 through the integrating of control and mKD embryos at dome and shield stages with setting control embryos as reference. To make the data comparable samples  we performed SCTransform normalization separately for each dataset. To further reduce the bais caused by sequencing depth of scRNA seq and percentage of mitochondrial genes  UMI variance and percent.mt were regressed out from SCTransform normalized data matrix by linear model with negative binomial distribution. All ChIP seq reads were aligned to the zebrafish reference genome danRer7 using Bowtie2 version 2.2.2 Langmead and Salzberg  2012 with the parameters –t –q –N 1 –L 25. All unmapped reads  non uniquely mapped reads and PCR duplicates were removed. For downstream analysis  the read counts were normalized by computing the numbers of reads per kilobase of bin per million of reads sequenced RPKM. To visualize the STAR ChIP seq signal in the UCSC genome browser  each read was extended by 250 bp and the coverage for each base was counted. Replicates of STAR ChIP seq were pooled for the downstream analysis. The ATAC seq data were mapped to the zebrafish reference genome danRer7 using Bowtie2 version 2.2.2 Langmead and Salzberg  2012 with the parameters –t –q –N 1 –L 25. All unmapped reads  non uniquely mapped reads and PCR duplicates were removed. For downstream analysis  the read counts were normalized by computing the numbers of reads per kilobase of bin per million of reads sequenced RPKM. To minimize the batch and cell type variation  RPKM values across whole genome were further Z score normalized. To visualize the CUT&RUN signals in the UCSC genome browser  each read was extended by 250 bp and the coverage for each base was counted. Genome build: danRer7 Supplementary files format and content: Matrix for scRNA seq data  gene expression table for total RNA seq data  Bigwig and narrowPeak for ChIP seq and ATAC seq data,embryo,Briefly  on the first day of OMIS Wu et al.  2018b  adult females at 5 mpf 12 mpf were anesthetized in 550 µg/ml tricaine Sigma  Cat A5040 in a petri dish. Then the fish was placed on a damp sponge with specific buffer 5.4 mM KCl  136.8 mM NaCl  4.2 mM NaHCO3  0.44 mM KH2PO4  0.25 mM Na2HPO4  and 0.5% wt/vol BSA. A cut was made on one side of belly to expose the ovary. The diluted MOs were microinjected into each oocyte. Rhodamine B Sigma  Cat R8881 was co injected with MOs as a dye. post injection  the wound on the belly was sewed with a surgical sewing needle carefully and quickly. Once the operation was done  the female was transferred into fish water supplemented with 32 µg/ml tricaine  10 unit/ml penicillin and 10 µg/ml streptomycin HyClone  Cat SV30010. Then the fish was transferred to fish water containing gradually reduced concentrations of tricaine. In the evening of the second day  the female was paired with a wild type male. In the morning of the third day  the pair started to chase and lay fertilized eggs naturally. The injected oocyte derived embryos were identified by co injected dye rhodamine B at very early developmental stages. The injection doses of dnmt1 MO were 5 ng per oocyte in the same assay. MOs were dissolved in RNase free water  and heated to 65°C for 11 min before microinjection.,Cell dissociation protocol was based on a previously described method Farrell et al.  2018 with modifications to adapt it for 10x Genomics platform. Briefly  dnmt1 mKD and control embryos were collected 20 min post fertilization and cultured as mentioned before. Then 15 embryos at dome or shield stages for each sample were transferred into plastic Petri dishes that had previously been coated with 2% agarose and soaked with DMEM/F12 media Gibco/Life Technologies  Cat 11330032 at least 2hrs. Next  embryos at desired stages were dechorionated and deyolked manually by forceps and transferred to a 1.5 ml Eppendorf tube with 50 µl DMEM/F12 media. Dissections were performed for up 15 min. The volume of DMEM/F12 meida containing embryos was adjusted to 200 µl  then cells were mechanically dissociated by flicking the tube 30 times and pipetting mixture 10 times through a 200 µl tip. The volume was adjusted to 1 ml with PBS containing 1.0% BSA  and spun to pellet cells at 300 g for 30s. The supernatant was removed and cells were resuspended in 80 µl of PBS containing 0.1% BSA and 20% Optiprep StemCell  Cat 07820  aiming for a concentration above 100 cells/µl. Cells were then passed through a cell sieve 100 µm for dome stage and 70 µm for shield stage. Single cell RNA seq library was performed with Chromium Next GEM Single Cell kit 10x Genomics  Cat PN 1000121 based on standard protocol. All libraries were sequenced by Illumina Hi Seq 1500 or 2500 or XTen platform according to manufacturer’s instruction.,The wild type AB strain was used in the experiments. The females post OMIS were fed with live adult brine shrimp thrice a day. All embryos were raised in Holtfreter’s solution at 28.5C and staged as described previously Kimmel et al.  1995.,strain:AB|genotype:WT|developmental stage:Dome|tissue:embryo|treatment:microinjection with Rhodamine B and dnmt1 MO at oogenesis III stage GV,GSM5351825,GSM5351825: Dome dnmt1 mKD scRNA seq rep2; Danio rerio; RNA Seq,GSM5351825,,1,Cell dissociation protocol was based on a previously described method Farrell et al.  2018 with modifications to adapt it for 10x Genomics platform. Briefly  dnmt1 mKD and control embryos were collected 20 min post fertilization and cultured as mentioned before. Then 15 embryos at dome or shield stages for each sample were transferred into plastic Petri dishes that had previously been coated with 2% agarose and soaked with DMEM/F12 media Gibco/Life Technologies  Cat 11330032 at least 2hrs. Next  embryos at desired stages were dechorionated and deyolked manually by forceps and transferred to a 1.5 ml Eppendorf tube with 50 µl DMEM/F12 media. Dissections were performed for up 15 min. The volume of DMEM/F12 meida containing embryos was adjusted to 200 µl  then cells were mechanically dissociated by flicking the tube 30 times and pipetting mixture 10 times through a 200 µl tip. The volume was adjusted to 1 ml with PBS containing 1.0% BSA  and spun to pellet cells at 300 g for 30s. The supernatant was removed and cells were resuspended in 80 µl of PBS containing 0.1% BSA and 20% Optiprep StemCell  Cat 07820  aiming for a concentration above 100 cells/µl. Cells were then passed through a cell sieve 100 µm for dome stage and 70 µm for shield stage. Single cell RNA seq library was performed with Chromium Next GEM Single Cell kit 10x Genomics  Cat PN 1000121 based on standard protocol. All libraries were sequenced by Illumina Hi Seq 1500 or 2500 or XTen platform according to manufacturer's instruction.,GEO Accession:GSM5351825,RNA-Seq,TRANSCRIPTOMIC,cDNA,PAIRED,ILLUMINA,Illumina HiSeq 1500,,SRP322195,,loader:fastq load.py,Dome_dnmt1_mKD_scRNA_seq_rep2_1_r1.fq.gz Dome_dnmt1_mKD_scRNA_seq_rep2_1_r2.fq.gz,fastq fastq,5239237200.0,17464124.0,GSM5351825 r1,0:150 1:150,A:1321051209;C:950485895;G:1001016620;T:1966497937;N:185539,150,150,,,1321051209,950485895,1001016620,1966497937,185539,SRX11041470,SRS9110398,SRA1239365,GEO,"THU-PKU Center for Life Sciences, Tsinghua University",2,0.33959,0.87634,0.0869,0.1399,0.95424,0.79011,0.57727,0.53102,150,150,T,B,mate1 technical by mapping diff,illumina,hiseq_era,unknown,cdna_unspecified,unknown,sc,single_cell_droplet,10x,,China,2021-06-01,Blastula,Embryo,Embryo Imprecise,All anatomical structures
64432,SRR14703459,SRX11041470,SRS9110398,SRP322195,PRJNA734348,Methylome inheritance and enhancer dememorization construct an epigenetic gate safeguarding embryonic programs,GSE175951,Other,Unlike that of mammals  the total DNA methylome of many cold blooded vertebrates is globally inherited from gametes to early embryos. In zebrafish  this is however accompanied by sweeping “dememorization” of enhancers prior to fertilization for sperm and just post fertilization for oocyte  as they undergo full methylation and are not demethylated again until phylotypic stage. The significance of both global methylome inheritance and enhancer dememorization in early embryos remains largely unknown. Adding to the puzzles  the zygotic mutant zebrafish of dnmt1  the major DNA methylation maintenance methyltransferase  surprisingly can develop to term. To solve the role of DNA methylation in early development  we generated zebrafish embryos derived from dnmt1 knocking down oocytes using a recently developed method OMIS microinjection in situ  which successfully eliminated DNA methylation before zygotic genome activation. dnmt1 deficient embryos failed to initiate epiboly and died around gastrulation. This is in part caused by activation of immune response and p53 regulated apoptosis  likely triggered by the derepression of transposable elements. Single cell RNA seq further revealed defective differentiation in these mutants. DNA methylation is also required for the establishment of repressive histone marks H3K27me3 and H2AK119ub. Strikingly  the loss of DNA methylation leads to extensive derepression of somatic genes and enhancers  which acquire ectopic H3K27ac  accessible chromatin  and H3K4me3. These somatic enhancers are preferentially CG rich and are bound by CG containing TFs. By contrast  embryonic enhancers are generally CG poor  methylation insensitive  and are bound by CG less TFs. Hence  the global DNA methylome inheritance is essential for vertebrate early development  and enhancer dememorization resets an epigenetic gate that separates embryonic and somatic programs. Overall design: By employing MethylC seq  total RNA seq  scRNA seq  CUT&RUN  ChIP seq  and ATAC seq in control and dnmt1 mKD embryos at oocyte  256 cell  dome and shield stages in zebrafish  we interrogated the function of DNA methylome and its inheritance in early zebrafish development.,,pubmed:34936444,,Dome dnmt1 mKD scRNA seq rep2,GSM5351825,,source name:embryo|strain:AB|genotype:WT|developmental stage:Dome|tissue:embryo|treatment:microinjection with Rhodamine B and dnmt1 MO at oogenesis III stage GV,Dome dnmt1 mKD scRNA seq rep2,All STEM seq datasets were mapped to the danRer7 reference genome by Bismark Krueger and Andrews  2011. Reads were trimmed with cutadaptor Martin  2011 using parameters:   minimum length 20   pair filter=any. Alignments were performed with the following parameters:  N 1  X 600   score min L 0  0.6. Multi mapped reads and PCR duplicates were removed. bismark methylation extractor was used to calculate the DNA methylation level. Total RNA seq data were firstly processed using Trim Galore! with default parameters to trim the adapter containing and low quality reads. The filtered data were then mapped to the zebrafish reference genome danRer7 by STAR version: STAR 2.5.3a modified Dobin et al.  2013 with the following parameters:   outSAMstrandField intronMotif   outSAMattributes All   outSAMunmapped Within   outSAMattrIHstart 0   outWigStrand Stranded   outFilterMultimapNmax 20   twopassMode Basic. The gene expression level was normalized to fragments per kilobase of transcript per million mapped FPKM values using Cufflinks version 2.2.1 Trapnell et al.  2012. The scRNA seq data were processed with Cell Ranger 3.1 for genome alignment danRer10  transcript counting  and gene cell barcode matrix generation. Before the downstream analysis  cells with unique detected genes lower than <2 000 and higher than 6 000 genes were discarded  and cells with high percentage of mitochondrial genes >5% were removed too. Subtypes of cells were identified with Seurat 3.0 through the integrating of control and mKD embryos at dome and shield stages with setting control embryos as reference. To make the data comparable samples  we performed SCTransform normalization separately for each dataset. To further reduce the bais caused by sequencing depth of scRNA seq and percentage of mitochondrial genes  UMI variance and percent.mt were regressed out from SCTransform normalized data matrix by linear model with negative binomial distribution. All ChIP seq reads were aligned to the zebrafish reference genome danRer7 using Bowtie2 version 2.2.2 Langmead and Salzberg  2012 with the parameters –t –q –N 1 –L 25. All unmapped reads  non uniquely mapped reads and PCR duplicates were removed. For downstream analysis  the read counts were normalized by computing the numbers of reads per kilobase of bin per million of reads sequenced RPKM. To visualize the STAR ChIP seq signal in the UCSC genome browser  each read was extended by 250 bp and the coverage for each base was counted. Replicates of STAR ChIP seq were pooled for the downstream analysis. The ATAC seq data were mapped to the zebrafish reference genome danRer7 using Bowtie2 version 2.2.2 Langmead and Salzberg  2012 with the parameters –t –q –N 1 –L 25. All unmapped reads  non uniquely mapped reads and PCR duplicates were removed. For downstream analysis  the read counts were normalized by computing the numbers of reads per kilobase of bin per million of reads sequenced RPKM. To minimize the batch and cell type variation  RPKM values across whole genome were further Z score normalized. To visualize the CUT&RUN signals in the UCSC genome browser  each read was extended by 250 bp and the coverage for each base was counted. Genome build: danRer7 Supplementary files format and content: Matrix for scRNA seq data  gene expression table for total RNA seq data  Bigwig and narrowPeak for ChIP seq and ATAC seq data,embryo,Briefly  on the first day of OMIS Wu et al.  2018b  adult females at 5 mpf 12 mpf were anesthetized in 550 µg/ml tricaine Sigma  Cat A5040 in a petri dish. Then the fish was placed on a damp sponge with specific buffer 5.4 mM KCl  136.8 mM NaCl  4.2 mM NaHCO3  0.44 mM KH2PO4  0.25 mM Na2HPO4  and 0.5% wt/vol BSA. A cut was made on one side of belly to expose the ovary. The diluted MOs were microinjected into each oocyte. Rhodamine B Sigma  Cat R8881 was co injected with MOs as a dye. post injection  the wound on the belly was sewed with a surgical sewing needle carefully and quickly. Once the operation was done  the female was transferred into fish water supplemented with 32 µg/ml tricaine  10 unit/ml penicillin and 10 µg/ml streptomycin HyClone  Cat SV30010. Then the fish was transferred to fish water containing gradually reduced concentrations of tricaine. In the evening of the second day  the female was paired with a wild type male. In the morning of the third day  the pair started to chase and lay fertilized eggs naturally. The injected oocyte derived embryos were identified by co injected dye rhodamine B at very early developmental stages. The injection doses of dnmt1 MO were 5 ng per oocyte in the same assay. MOs were dissolved in RNase free water  and heated to 65°C for 11 min before microinjection.,Cell dissociation protocol was based on a previously described method Farrell et al.  2018 with modifications to adapt it for 10x Genomics platform. Briefly  dnmt1 mKD and control embryos were collected 20 min post fertilization and cultured as mentioned before. Then 15 embryos at dome or shield stages for each sample were transferred into plastic Petri dishes that had previously been coated with 2% agarose and soaked with DMEM/F12 media Gibco/Life Technologies  Cat 11330032 at least 2hrs. Next  embryos at desired stages were dechorionated and deyolked manually by forceps and transferred to a 1.5 ml Eppendorf tube with 50 µl DMEM/F12 media. Dissections were performed for up 15 min. The volume of DMEM/F12 meida containing embryos was adjusted to 200 µl  then cells were mechanically dissociated by flicking the tube 30 times and pipetting mixture 10 times through a 200 µl tip. The volume was adjusted to 1 ml with PBS containing 1.0% BSA  and spun to pellet cells at 300 g for 30s. The supernatant was removed and cells were resuspended in 80 µl of PBS containing 0.1% BSA and 20% Optiprep StemCell  Cat 07820  aiming for a concentration above 100 cells/µl. Cells were then passed through a cell sieve 100 µm for dome stage and 70 µm for shield stage. Single cell RNA seq library was performed with Chromium Next GEM Single Cell kit 10x Genomics  Cat PN 1000121 based on standard protocol. All libraries were sequenced by Illumina Hi Seq 1500 or 2500 or XTen platform according to manufacturer’s instruction.,The wild type AB strain was used in the experiments. The females post OMIS were fed with live adult brine shrimp thrice a day. All embryos were raised in Holtfreter’s solution at 28.5C and staged as described previously Kimmel et al.  1995.,strain:AB|genotype:WT|developmental stage:Dome|tissue:embryo|treatment:microinjection with Rhodamine B and dnmt1 MO at oogenesis III stage GV,GSM5351825,GSM5351825: Dome dnmt1 mKD scRNA seq rep2; Danio rerio; RNA Seq,GSM5351825,,1,Cell dissociation protocol was based on a previously described method Farrell et al.  2018 with modifications to adapt it for 10x Genomics platform. Briefly  dnmt1 mKD and control embryos were collected 20 min post fertilization and cultured as mentioned before. Then 15 embryos at dome or shield stages for each sample were transferred into plastic Petri dishes that had previously been coated with 2% agarose and soaked with DMEM/F12 media Gibco/Life Technologies  Cat 11330032 at least 2hrs. Next  embryos at desired stages were dechorionated and deyolked manually by forceps and transferred to a 1.5 ml Eppendorf tube with 50 µl DMEM/F12 media. Dissections were performed for up 15 min. The volume of DMEM/F12 meida containing embryos was adjusted to 200 µl  then cells were mechanically dissociated by flicking the tube 30 times and pipetting mixture 10 times through a 200 µl tip. The volume was adjusted to 1 ml with PBS containing 1.0% BSA  and spun to pellet cells at 300 g for 30s. The supernatant was removed and cells were resuspended in 80 µl of PBS containing 0.1% BSA and 20% Optiprep StemCell  Cat 07820  aiming for a concentration above 100 cells/µl. Cells were then passed through a cell sieve 100 µm for dome stage and 70 µm for shield stage. Single cell RNA seq library was performed with Chromium Next GEM Single Cell kit 10x Genomics  Cat PN 1000121 based on standard protocol. All libraries were sequenced by Illumina Hi Seq 1500 or 2500 or XTen platform according to manufacturer's instruction.,GEO Accession:GSM5351825,RNA-Seq,TRANSCRIPTOMIC,cDNA,PAIRED,ILLUMINA,Illumina HiSeq 1500,,SRP322195,,loader:fastq load.py,Dome_dnmt1_mKD_scRNA_seq_rep2_2_r1.fq.gz Dome_dnmt1_mKD_scRNA_seq_rep2_2_r2.fq.gz,fastq fastq,4906402200.0,16354674.0,GSM5351825 r2,0:150 1:150,A:1236945672;C:891422080;G:938344729;T:1839517198;N:172521,150,150,,,1236945672,891422080,938344729,1839517198,172521,SRX11041470,SRS9110398,SRA1239365,GEO,"THU-PKU Center for Life Sciences, Tsinghua University",2,0.34175,0.87674,0.08978,0.13862,0.95607,0.79022,0.58694,0.53142,150,150,T,B,mate1 technical by mapping diff,illumina,hiseq_era,unknown,cdna_unspecified,unknown,sc,single_cell_droplet,10x,,China,2021-06-01,Blastula,Embryo,Embryo Imprecise,All anatomical structures
64433,SRR14703460,SRX11041470,SRS9110398,SRP322195,PRJNA734348,Methylome inheritance and enhancer dememorization construct an epigenetic gate safeguarding embryonic programs,GSE175951,Other,Unlike that of mammals  the total DNA methylome of many cold blooded vertebrates is globally inherited from gametes to early embryos. In zebrafish  this is however accompanied by sweeping “dememorization” of enhancers prior to fertilization for sperm and just post fertilization for oocyte  as they undergo full methylation and are not demethylated again until phylotypic stage. The significance of both global methylome inheritance and enhancer dememorization in early embryos remains largely unknown. Adding to the puzzles  the zygotic mutant zebrafish of dnmt1  the major DNA methylation maintenance methyltransferase  surprisingly can develop to term. To solve the role of DNA methylation in early development  we generated zebrafish embryos derived from dnmt1 knocking down oocytes using a recently developed method OMIS microinjection in situ  which successfully eliminated DNA methylation before zygotic genome activation. dnmt1 deficient embryos failed to initiate epiboly and died around gastrulation. This is in part caused by activation of immune response and p53 regulated apoptosis  likely triggered by the derepression of transposable elements. Single cell RNA seq further revealed defective differentiation in these mutants. DNA methylation is also required for the establishment of repressive histone marks H3K27me3 and H2AK119ub. Strikingly  the loss of DNA methylation leads to extensive derepression of somatic genes and enhancers  which acquire ectopic H3K27ac  accessible chromatin  and H3K4me3. These somatic enhancers are preferentially CG rich and are bound by CG containing TFs. By contrast  embryonic enhancers are generally CG poor  methylation insensitive  and are bound by CG less TFs. Hence  the global DNA methylome inheritance is essential for vertebrate early development  and enhancer dememorization resets an epigenetic gate that separates embryonic and somatic programs. Overall design: By employing MethylC seq  total RNA seq  scRNA seq  CUT&RUN  ChIP seq  and ATAC seq in control and dnmt1 mKD embryos at oocyte  256 cell  dome and shield stages in zebrafish  we interrogated the function of DNA methylome and its inheritance in early zebrafish development.,,pubmed:34936444,,Dome dnmt1 mKD scRNA seq rep2,GSM5351825,,source name:embryo|strain:AB|genotype:WT|developmental stage:Dome|tissue:embryo|treatment:microinjection with Rhodamine B and dnmt1 MO at oogenesis III stage GV,Dome dnmt1 mKD scRNA seq rep2,All STEM seq datasets were mapped to the danRer7 reference genome by Bismark Krueger and Andrews  2011. Reads were trimmed with cutadaptor Martin  2011 using parameters:   minimum length 20   pair filter=any. Alignments were performed with the following parameters:  N 1  X 600   score min L 0  0.6. Multi mapped reads and PCR duplicates were removed. bismark methylation extractor was used to calculate the DNA methylation level. Total RNA seq data were firstly processed using Trim Galore! with default parameters to trim the adapter containing and low quality reads. The filtered data were then mapped to the zebrafish reference genome danRer7 by STAR version: STAR 2.5.3a modified Dobin et al.  2013 with the following parameters:   outSAMstrandField intronMotif   outSAMattributes All   outSAMunmapped Within   outSAMattrIHstart 0   outWigStrand Stranded   outFilterMultimapNmax 20   twopassMode Basic. The gene expression level was normalized to fragments per kilobase of transcript per million mapped FPKM values using Cufflinks version 2.2.1 Trapnell et al.  2012. The scRNA seq data were processed with Cell Ranger 3.1 for genome alignment danRer10  transcript counting  and gene cell barcode matrix generation. Before the downstream analysis  cells with unique detected genes lower than <2 000 and higher than 6 000 genes were discarded  and cells with high percentage of mitochondrial genes >5% were removed too. Subtypes of cells were identified with Seurat 3.0 through the integrating of control and mKD embryos at dome and shield stages with setting control embryos as reference. To make the data comparable samples  we performed SCTransform normalization separately for each dataset. To further reduce the bais caused by sequencing depth of scRNA seq and percentage of mitochondrial genes  UMI variance and percent.mt were regressed out from SCTransform normalized data matrix by linear model with negative binomial distribution. All ChIP seq reads were aligned to the zebrafish reference genome danRer7 using Bowtie2 version 2.2.2 Langmead and Salzberg  2012 with the parameters –t –q –N 1 –L 25. All unmapped reads  non uniquely mapped reads and PCR duplicates were removed. For downstream analysis  the read counts were normalized by computing the numbers of reads per kilobase of bin per million of reads sequenced RPKM. To visualize the STAR ChIP seq signal in the UCSC genome browser  each read was extended by 250 bp and the coverage for each base was counted. Replicates of STAR ChIP seq were pooled for the downstream analysis. The ATAC seq data were mapped to the zebrafish reference genome danRer7 using Bowtie2 version 2.2.2 Langmead and Salzberg  2012 with the parameters –t –q –N 1 –L 25. All unmapped reads  non uniquely mapped reads and PCR duplicates were removed. For downstream analysis  the read counts were normalized by computing the numbers of reads per kilobase of bin per million of reads sequenced RPKM. To minimize the batch and cell type variation  RPKM values across whole genome were further Z score normalized. To visualize the CUT&RUN signals in the UCSC genome browser  each read was extended by 250 bp and the coverage for each base was counted. Genome build: danRer7 Supplementary files format and content: Matrix for scRNA seq data  gene expression table for total RNA seq data  Bigwig and narrowPeak for ChIP seq and ATAC seq data,embryo,Briefly  on the first day of OMIS Wu et al.  2018b  adult females at 5 mpf 12 mpf were anesthetized in 550 µg/ml tricaine Sigma  Cat A5040 in a petri dish. Then the fish was placed on a damp sponge with specific buffer 5.4 mM KCl  136.8 mM NaCl  4.2 mM NaHCO3  0.44 mM KH2PO4  0.25 mM Na2HPO4  and 0.5% wt/vol BSA. A cut was made on one side of belly to expose the ovary. The diluted MOs were microinjected into each oocyte. Rhodamine B Sigma  Cat R8881 was co injected with MOs as a dye. post injection  the wound on the belly was sewed with a surgical sewing needle carefully and quickly. Once the operation was done  the female was transferred into fish water supplemented with 32 µg/ml tricaine  10 unit/ml penicillin and 10 µg/ml streptomycin HyClone  Cat SV30010. Then the fish was transferred to fish water containing gradually reduced concentrations of tricaine. In the evening of the second day  the female was paired with a wild type male. In the morning of the third day  the pair started to chase and lay fertilized eggs naturally. The injected oocyte derived embryos were identified by co injected dye rhodamine B at very early developmental stages. The injection doses of dnmt1 MO were 5 ng per oocyte in the same assay. MOs were dissolved in RNase free water  and heated to 65°C for 11 min before microinjection.,Cell dissociation protocol was based on a previously described method Farrell et al.  2018 with modifications to adapt it for 10x Genomics platform. Briefly  dnmt1 mKD and control embryos were collected 20 min post fertilization and cultured as mentioned before. Then 15 embryos at dome or shield stages for each sample were transferred into plastic Petri dishes that had previously been coated with 2% agarose and soaked with DMEM/F12 media Gibco/Life Technologies  Cat 11330032 at least 2hrs. Next  embryos at desired stages were dechorionated and deyolked manually by forceps and transferred to a 1.5 ml Eppendorf tube with 50 µl DMEM/F12 media. Dissections were performed for up 15 min. The volume of DMEM/F12 meida containing embryos was adjusted to 200 µl  then cells were mechanically dissociated by flicking the tube 30 times and pipetting mixture 10 times through a 200 µl tip. The volume was adjusted to 1 ml with PBS containing 1.0% BSA  and spun to pellet cells at 300 g for 30s. The supernatant was removed and cells were resuspended in 80 µl of PBS containing 0.1% BSA and 20% Optiprep StemCell  Cat 07820  aiming for a concentration above 100 cells/µl. Cells were then passed through a cell sieve 100 µm for dome stage and 70 µm for shield stage. Single cell RNA seq library was performed with Chromium Next GEM Single Cell kit 10x Genomics  Cat PN 1000121 based on standard protocol. All libraries were sequenced by Illumina Hi Seq 1500 or 2500 or XTen platform according to manufacturer’s instruction.,The wild type AB strain was used in the experiments. The females post OMIS were fed with live adult brine shrimp thrice a day. All embryos were raised in Holtfreter’s solution at 28.5C and staged as described previously Kimmel et al.  1995.,strain:AB|genotype:WT|developmental stage:Dome|tissue:embryo|treatment:microinjection with Rhodamine B and dnmt1 MO at oogenesis III stage GV,GSM5351825,GSM5351825: Dome dnmt1 mKD scRNA seq rep2; Danio rerio; RNA Seq,GSM5351825,,1,Cell dissociation protocol was based on a previously described method Farrell et al.  2018 with modifications to adapt it for 10x Genomics platform. Briefly  dnmt1 mKD and control embryos were collected 20 min post fertilization and cultured as mentioned before. Then 15 embryos at dome or shield stages for each sample were transferred into plastic Petri dishes that had previously been coated with 2% agarose and soaked with DMEM/F12 media Gibco/Life Technologies  Cat 11330032 at least 2hrs. Next  embryos at desired stages were dechorionated and deyolked manually by forceps and transferred to a 1.5 ml Eppendorf tube with 50 µl DMEM/F12 media. Dissections were performed for up 15 min. The volume of DMEM/F12 meida containing embryos was adjusted to 200 µl  then cells were mechanically dissociated by flicking the tube 30 times and pipetting mixture 10 times through a 200 µl tip. The volume was adjusted to 1 ml with PBS containing 1.0% BSA  and spun to pellet cells at 300 g for 30s. The supernatant was removed and cells were resuspended in 80 µl of PBS containing 0.1% BSA and 20% Optiprep StemCell  Cat 07820  aiming for a concentration above 100 cells/µl. Cells were then passed through a cell sieve 100 µm for dome stage and 70 µm for shield stage. Single cell RNA seq library was performed with Chromium Next GEM Single Cell kit 10x Genomics  Cat PN 1000121 based on standard protocol. All libraries were sequenced by Illumina Hi Seq 1500 or 2500 or XTen platform according to manufacturer's instruction.,GEO Accession:GSM5351825,RNA-Seq,TRANSCRIPTOMIC,cDNA,PAIRED,ILLUMINA,Illumina HiSeq 1500,,SRP322195,,loader:fastq load.py,Dome_dnmt1_mKD_scRNA_seq_rep2_3_r1.fq.gz Dome_dnmt1_mKD_scRNA_seq_rep2_3_r2.fq.gz,fastq fastq,4989781200.0,16632604.0,GSM5351825 r3,0:150 1:150,A:1259695012;C:902892681;G:950375679;T:1876642673;N:175155,150,150,,,1259695012,902892681,950375679,1876642673,175155,SRX11041470,SRS9110398,SRA1239365,GEO,"THU-PKU Center for Life Sciences, Tsinghua University",2,0.34667,0.87702,0.09176,0.14046,0.95396,0.78837,0.58525,0.53456,150,150,T,B,mate1 technical by mapping diff,illumina,hiseq_era,unknown,cdna_unspecified,unknown,sc,single_cell_droplet,10x,,China,2021-06-01,Blastula,Embryo,Embryo Imprecise,All anatomical structures
64434,SRR14703461,SRX11041470,SRS9110398,SRP322195,PRJNA734348,Methylome inheritance and enhancer dememorization construct an epigenetic gate safeguarding embryonic programs,GSE175951,Other,Unlike that of mammals  the total DNA methylome of many cold blooded vertebrates is globally inherited from gametes to early embryos. In zebrafish  this is however accompanied by sweeping “dememorization” of enhancers prior to fertilization for sperm and just post fertilization for oocyte  as they undergo full methylation and are not demethylated again until phylotypic stage. The significance of both global methylome inheritance and enhancer dememorization in early embryos remains largely unknown. Adding to the puzzles  the zygotic mutant zebrafish of dnmt1  the major DNA methylation maintenance methyltransferase  surprisingly can develop to term. To solve the role of DNA methylation in early development  we generated zebrafish embryos derived from dnmt1 knocking down oocytes using a recently developed method OMIS microinjection in situ  which successfully eliminated DNA methylation before zygotic genome activation. dnmt1 deficient embryos failed to initiate epiboly and died around gastrulation. This is in part caused by activation of immune response and p53 regulated apoptosis  likely triggered by the derepression of transposable elements. Single cell RNA seq further revealed defective differentiation in these mutants. DNA methylation is also required for the establishment of repressive histone marks H3K27me3 and H2AK119ub. Strikingly  the loss of DNA methylation leads to extensive derepression of somatic genes and enhancers  which acquire ectopic H3K27ac  accessible chromatin  and H3K4me3. These somatic enhancers are preferentially CG rich and are bound by CG containing TFs. By contrast  embryonic enhancers are generally CG poor  methylation insensitive  and are bound by CG less TFs. Hence  the global DNA methylome inheritance is essential for vertebrate early development  and enhancer dememorization resets an epigenetic gate that separates embryonic and somatic programs. Overall design: By employing MethylC seq  total RNA seq  scRNA seq  CUT&RUN  ChIP seq  and ATAC seq in control and dnmt1 mKD embryos at oocyte  256 cell  dome and shield stages in zebrafish  we interrogated the function of DNA methylome and its inheritance in early zebrafish development.,,pubmed:34936444,,Dome dnmt1 mKD scRNA seq rep2,GSM5351825,,source name:embryo|strain:AB|genotype:WT|developmental stage:Dome|tissue:embryo|treatment:microinjection with Rhodamine B and dnmt1 MO at oogenesis III stage GV,Dome dnmt1 mKD scRNA seq rep2,All STEM seq datasets were mapped to the danRer7 reference genome by Bismark Krueger and Andrews  2011. Reads were trimmed with cutadaptor Martin  2011 using parameters:   minimum length 20   pair filter=any. Alignments were performed with the following parameters:  N 1  X 600   score min L 0  0.6. Multi mapped reads and PCR duplicates were removed. bismark methylation extractor was used to calculate the DNA methylation level. Total RNA seq data were firstly processed using Trim Galore! with default parameters to trim the adapter containing and low quality reads. The filtered data were then mapped to the zebrafish reference genome danRer7 by STAR version: STAR 2.5.3a modified Dobin et al.  2013 with the following parameters:   outSAMstrandField intronMotif   outSAMattributes All   outSAMunmapped Within   outSAMattrIHstart 0   outWigStrand Stranded   outFilterMultimapNmax 20   twopassMode Basic. The gene expression level was normalized to fragments per kilobase of transcript per million mapped FPKM values using Cufflinks version 2.2.1 Trapnell et al.  2012. The scRNA seq data were processed with Cell Ranger 3.1 for genome alignment danRer10  transcript counting  and gene cell barcode matrix generation. Before the downstream analysis  cells with unique detected genes lower than <2 000 and higher than 6 000 genes were discarded  and cells with high percentage of mitochondrial genes >5% were removed too. Subtypes of cells were identified with Seurat 3.0 through the integrating of control and mKD embryos at dome and shield stages with setting control embryos as reference. To make the data comparable samples  we performed SCTransform normalization separately for each dataset. To further reduce the bais caused by sequencing depth of scRNA seq and percentage of mitochondrial genes  UMI variance and percent.mt were regressed out from SCTransform normalized data matrix by linear model with negative binomial distribution. All ChIP seq reads were aligned to the zebrafish reference genome danRer7 using Bowtie2 version 2.2.2 Langmead and Salzberg  2012 with the parameters –t –q –N 1 –L 25. All unmapped reads  non uniquely mapped reads and PCR duplicates were removed. For downstream analysis  the read counts were normalized by computing the numbers of reads per kilobase of bin per million of reads sequenced RPKM. To visualize the STAR ChIP seq signal in the UCSC genome browser  each read was extended by 250 bp and the coverage for each base was counted. Replicates of STAR ChIP seq were pooled for the downstream analysis. The ATAC seq data were mapped to the zebrafish reference genome danRer7 using Bowtie2 version 2.2.2 Langmead and Salzberg  2012 with the parameters –t –q –N 1 –L 25. All unmapped reads  non uniquely mapped reads and PCR duplicates were removed. For downstream analysis  the read counts were normalized by computing the numbers of reads per kilobase of bin per million of reads sequenced RPKM. To minimize the batch and cell type variation  RPKM values across whole genome were further Z score normalized. To visualize the CUT&RUN signals in the UCSC genome browser  each read was extended by 250 bp and the coverage for each base was counted. Genome build: danRer7 Supplementary files format and content: Matrix for scRNA seq data  gene expression table for total RNA seq data  Bigwig and narrowPeak for ChIP seq and ATAC seq data,embryo,Briefly  on the first day of OMIS Wu et al.  2018b  adult females at 5 mpf 12 mpf were anesthetized in 550 µg/ml tricaine Sigma  Cat A5040 in a petri dish. Then the fish was placed on a damp sponge with specific buffer 5.4 mM KCl  136.8 mM NaCl  4.2 mM NaHCO3  0.44 mM KH2PO4  0.25 mM Na2HPO4  and 0.5% wt/vol BSA. A cut was made on one side of belly to expose the ovary. The diluted MOs were microinjected into each oocyte. Rhodamine B Sigma  Cat R8881 was co injected with MOs as a dye. post injection  the wound on the belly was sewed with a surgical sewing needle carefully and quickly. Once the operation was done  the female was transferred into fish water supplemented with 32 µg/ml tricaine  10 unit/ml penicillin and 10 µg/ml streptomycin HyClone  Cat SV30010. Then the fish was transferred to fish water containing gradually reduced concentrations of tricaine. In the evening of the second day  the female was paired with a wild type male. In the morning of the third day  the pair started to chase and lay fertilized eggs naturally. The injected oocyte derived embryos were identified by co injected dye rhodamine B at very early developmental stages. The injection doses of dnmt1 MO were 5 ng per oocyte in the same assay. MOs were dissolved in RNase free water  and heated to 65°C for 11 min before microinjection.,Cell dissociation protocol was based on a previously described method Farrell et al.  2018 with modifications to adapt it for 10x Genomics platform. Briefly  dnmt1 mKD and control embryos were collected 20 min post fertilization and cultured as mentioned before. Then 15 embryos at dome or shield stages for each sample were transferred into plastic Petri dishes that had previously been coated with 2% agarose and soaked with DMEM/F12 media Gibco/Life Technologies  Cat 11330032 at least 2hrs. Next  embryos at desired stages were dechorionated and deyolked manually by forceps and transferred to a 1.5 ml Eppendorf tube with 50 µl DMEM/F12 media. Dissections were performed for up 15 min. The volume of DMEM/F12 meida containing embryos was adjusted to 200 µl  then cells were mechanically dissociated by flicking the tube 30 times and pipetting mixture 10 times through a 200 µl tip. The volume was adjusted to 1 ml with PBS containing 1.0% BSA  and spun to pellet cells at 300 g for 30s. The supernatant was removed and cells were resuspended in 80 µl of PBS containing 0.1% BSA and 20% Optiprep StemCell  Cat 07820  aiming for a concentration above 100 cells/µl. Cells were then passed through a cell sieve 100 µm for dome stage and 70 µm for shield stage. Single cell RNA seq library was performed with Chromium Next GEM Single Cell kit 10x Genomics  Cat PN 1000121 based on standard protocol. All libraries were sequenced by Illumina Hi Seq 1500 or 2500 or XTen platform according to manufacturer’s instruction.,The wild type AB strain was used in the experiments. The females post OMIS were fed with live adult brine shrimp thrice a day. All embryos were raised in Holtfreter’s solution at 28.5C and staged as described previously Kimmel et al.  1995.,strain:AB|genotype:WT|developmental stage:Dome|tissue:embryo|treatment:microinjection with Rhodamine B and dnmt1 MO at oogenesis III stage GV,GSM5351825,GSM5351825: Dome dnmt1 mKD scRNA seq rep2; Danio rerio; RNA Seq,GSM5351825,,1,Cell dissociation protocol was based on a previously described method Farrell et al.  2018 with modifications to adapt it for 10x Genomics platform. Briefly  dnmt1 mKD and control embryos were collected 20 min post fertilization and cultured as mentioned before. Then 15 embryos at dome or shield stages for each sample were transferred into plastic Petri dishes that had previously been coated with 2% agarose and soaked with DMEM/F12 media Gibco/Life Technologies  Cat 11330032 at least 2hrs. Next  embryos at desired stages were dechorionated and deyolked manually by forceps and transferred to a 1.5 ml Eppendorf tube with 50 µl DMEM/F12 media. Dissections were performed for up 15 min. The volume of DMEM/F12 meida containing embryos was adjusted to 200 µl  then cells were mechanically dissociated by flicking the tube 30 times and pipetting mixture 10 times through a 200 µl tip. The volume was adjusted to 1 ml with PBS containing 1.0% BSA  and spun to pellet cells at 300 g for 30s. The supernatant was removed and cells were resuspended in 80 µl of PBS containing 0.1% BSA and 20% Optiprep StemCell  Cat 07820  aiming for a concentration above 100 cells/µl. Cells were then passed through a cell sieve 100 µm for dome stage and 70 µm for shield stage. Single cell RNA seq library was performed with Chromium Next GEM Single Cell kit 10x Genomics  Cat PN 1000121 based on standard protocol. All libraries were sequenced by Illumina Hi Seq 1500 or 2500 or XTen platform according to manufacturer's instruction.,GEO Accession:GSM5351825,RNA-Seq,TRANSCRIPTOMIC,cDNA,PAIRED,ILLUMINA,Illumina HiSeq 1500,,SRP322195,,loader:fastq load.py,Dome_dnmt1_mKD_scRNA_seq_rep2_4_r1.fq.gz Dome_dnmt1_mKD_scRNA_seq_rep2_4_r2.fq.gz,fastq fastq,4380423600.0,14601412.0,GSM5351825 r4,0:150 1:150,A:1101017284;C:795734654;G:826725666;T:1656795538;N:150458,150,150,,,1101017284,795734654,826725666,1656795538,150458,SRX11041470,SRS9110398,SRA1239365,GEO,"THU-PKU Center for Life Sciences, Tsinghua University",2,0.3746,0.87751,0.09966,0.13892,0.95668,0.78926,0.57333,0.52678,150,150,B,B,mate1-mate2 similar by mapping diff,illumina,hiseq_era,unknown,cdna_unspecified,unknown,sc,single_cell_droplet,10x,,China,2021-06-01,Blastula,Embryo,Embryo Imprecise,All anatomical structures
64435,SRR14703454,SRX11041469,SRS9110396,SRP322195,PRJNA734348,Methylome inheritance and enhancer dememorization construct an epigenetic gate safeguarding embryonic programs,GSE175951,Other,Unlike that of mammals  the total DNA methylome of many cold blooded vertebrates is globally inherited from gametes to early embryos. In zebrafish  this is however accompanied by sweeping “dememorization” of enhancers prior to fertilization for sperm and just post fertilization for oocyte  as they undergo full methylation and are not demethylated again until phylotypic stage. The significance of both global methylome inheritance and enhancer dememorization in early embryos remains largely unknown. Adding to the puzzles  the zygotic mutant zebrafish of dnmt1  the major DNA methylation maintenance methyltransferase  surprisingly can develop to term. To solve the role of DNA methylation in early development  we generated zebrafish embryos derived from dnmt1 knocking down oocytes using a recently developed method OMIS microinjection in situ  which successfully eliminated DNA methylation before zygotic genome activation. dnmt1 deficient embryos failed to initiate epiboly and died around gastrulation. This is in part caused by activation of immune response and p53 regulated apoptosis  likely triggered by the derepression of transposable elements. Single cell RNA seq further revealed defective differentiation in these mutants. DNA methylation is also required for the establishment of repressive histone marks H3K27me3 and H2AK119ub. Strikingly  the loss of DNA methylation leads to extensive derepression of somatic genes and enhancers  which acquire ectopic H3K27ac  accessible chromatin  and H3K4me3. These somatic enhancers are preferentially CG rich and are bound by CG containing TFs. By contrast  embryonic enhancers are generally CG poor  methylation insensitive  and are bound by CG less TFs. Hence  the global DNA methylome inheritance is essential for vertebrate early development  and enhancer dememorization resets an epigenetic gate that separates embryonic and somatic programs. Overall design: By employing MethylC seq  total RNA seq  scRNA seq  CUT&RUN  ChIP seq  and ATAC seq in control and dnmt1 mKD embryos at oocyte  256 cell  dome and shield stages in zebrafish  we interrogated the function of DNA methylome and its inheritance in early zebrafish development.,,pubmed:34936444,,Dome dnmt1 mKD scRNA seq rep1,GSM5351824,,source name:embryo|strain:AB|genotype:WT|developmental stage:Dome|tissue:embryo|treatment:microinjection with Rhodamine B and dnmt1 MO at oogenesis III stage GV,Dome dnmt1 mKD scRNA seq rep1,All STEM seq datasets were mapped to the danRer7 reference genome by Bismark Krueger and Andrews  2011. Reads were trimmed with cutadaptor Martin  2011 using parameters:   minimum length 20   pair filter=any. Alignments were performed with the following parameters:  N 1  X 600   score min L 0  0.6. Multi mapped reads and PCR duplicates were removed. bismark methylation extractor was used to calculate the DNA methylation level. Total RNA seq data were firstly processed using Trim Galore! with default parameters to trim the adapter containing and low quality reads. The filtered data were then mapped to the zebrafish reference genome danRer7 by STAR version: STAR 2.5.3a modified Dobin et al.  2013 with the following parameters:   outSAMstrandField intronMotif   outSAMattributes All   outSAMunmapped Within   outSAMattrIHstart 0   outWigStrand Stranded   outFilterMultimapNmax 20   twopassMode Basic. The gene expression level was normalized to fragments per kilobase of transcript per million mapped FPKM values using Cufflinks version 2.2.1 Trapnell et al.  2012. The scRNA seq data were processed with Cell Ranger 3.1 for genome alignment danRer10  transcript counting  and gene cell barcode matrix generation. Before the downstream analysis  cells with unique detected genes lower than <2 000 and higher than 6 000 genes were discarded  and cells with high percentage of mitochondrial genes >5% were removed too. Subtypes of cells were identified with Seurat 3.0 through the integrating of control and mKD embryos at dome and shield stages with setting control embryos as reference. To make the data comparable samples  we performed SCTransform normalization separately for each dataset. To further reduce the bais caused by sequencing depth of scRNA seq and percentage of mitochondrial genes  UMI variance and percent.mt were regressed out from SCTransform normalized data matrix by linear model with negative binomial distribution. All ChIP seq reads were aligned to the zebrafish reference genome danRer7 using Bowtie2 version 2.2.2 Langmead and Salzberg  2012 with the parameters –t –q –N 1 –L 25. All unmapped reads  non uniquely mapped reads and PCR duplicates were removed. For downstream analysis  the read counts were normalized by computing the numbers of reads per kilobase of bin per million of reads sequenced RPKM. To visualize the STAR ChIP seq signal in the UCSC genome browser  each read was extended by 250 bp and the coverage for each base was counted. Replicates of STAR ChIP seq were pooled for the downstream analysis. The ATAC seq data were mapped to the zebrafish reference genome danRer7 using Bowtie2 version 2.2.2 Langmead and Salzberg  2012 with the parameters –t –q –N 1 –L 25. All unmapped reads  non uniquely mapped reads and PCR duplicates were removed. For downstream analysis  the read counts were normalized by computing the numbers of reads per kilobase of bin per million of reads sequenced RPKM. To minimize the batch and cell type variation  RPKM values across whole genome were further Z score normalized. To visualize the CUT&RUN signals in the UCSC genome browser  each read was extended by 250 bp and the coverage for each base was counted. Genome build: danRer7 Supplementary files format and content: Matrix for scRNA seq data  gene expression table for total RNA seq data  Bigwig and narrowPeak for ChIP seq and ATAC seq data,embryo,Briefly  on the first day of OMIS Wu et al.  2018b  adult females at 5 mpf 12 mpf were anesthetized in 550 µg/ml tricaine Sigma  Cat A5040 in a petri dish. Then the fish was placed on a damp sponge with specific buffer 5.4 mM KCl  136.8 mM NaCl  4.2 mM NaHCO3  0.44 mM KH2PO4  0.25 mM Na2HPO4  and 0.5% wt/vol BSA. A cut was made on one side of belly to expose the ovary. The diluted MOs were microinjected into each oocyte. Rhodamine B Sigma  Cat R8881 was co injected with MOs as a dye. post injection  the wound on the belly was sewed with a surgical sewing needle carefully and quickly. Once the operation was done  the female was transferred into fish water supplemented with 32 µg/ml tricaine  10 unit/ml penicillin and 10 µg/ml streptomycin HyClone  Cat SV30010. Then the fish was transferred to fish water containing gradually reduced concentrations of tricaine. In the evening of the second day  the female was paired with a wild type male. In the morning of the third day  the pair started to chase and lay fertilized eggs naturally. The injected oocyte derived embryos were identified by co injected dye rhodamine B at very early developmental stages. The injection doses of dnmt1 MO were 5 ng per oocyte in the same assay. MOs were dissolved in RNase free water  and heated to 65°C for 11 min before microinjection.,Cell dissociation protocol was based on a previously described method Farrell et al.  2018 with modifications to adapt it for 10x Genomics platform. Briefly  dnmt1 mKD and control embryos were collected 20 min post fertilization and cultured as mentioned before. Then 15 embryos at dome or shield stages for each sample were transferred into plastic Petri dishes that had previously been coated with 2% agarose and soaked with DMEM/F12 media Gibco/Life Technologies  Cat 11330032 at least 2hrs. Next  embryos at desired stages were dechorionated and deyolked manually by forceps and transferred to a 1.5 ml Eppendorf tube with 50 µl DMEM/F12 media. Dissections were performed for up 15 min. The volume of DMEM/F12 meida containing embryos was adjusted to 200 µl  then cells were mechanically dissociated by flicking the tube 30 times and pipetting mixture 10 times through a 200 µl tip. The volume was adjusted to 1 ml with PBS containing 1.0% BSA  and spun to pellet cells at 300 g for 30s. The supernatant was removed and cells were resuspended in 80 µl of PBS containing 0.1% BSA and 20% Optiprep StemCell  Cat 07820  aiming for a concentration above 100 cells/µl. Cells were then passed through a cell sieve 100 µm for dome stage and 70 µm for shield stage. Single cell RNA seq library was performed with Chromium Next GEM Single Cell kit 10x Genomics  Cat PN 1000121 based on standard protocol. All libraries were sequenced by Illumina Hi Seq 1500 or 2500 or XTen platform according to manufacturer’s instruction.,The wild type AB strain was used in the experiments. The females post OMIS were fed with live adult brine shrimp thrice a day. All embryos were raised in Holtfreter’s solution at 28.5C and staged as described previously Kimmel et al.  1995.,strain:AB|genotype:WT|developmental stage:Dome|tissue:embryo|treatment:microinjection with Rhodamine B and dnmt1 MO at oogenesis III stage GV,GSM5351824,GSM5351824: Dome dnmt1 mKD scRNA seq rep1; Danio rerio; RNA Seq,GSM5351824,,1,Cell dissociation protocol was based on a previously described method Farrell et al.  2018 with modifications to adapt it for 10x Genomics platform. Briefly  dnmt1 mKD and control embryos were collected 20 min post fertilization and cultured as mentioned before. Then 15 embryos at dome or shield stages for each sample were transferred into plastic Petri dishes that had previously been coated with 2% agarose and soaked with DMEM/F12 media Gibco/Life Technologies  Cat 11330032 at least 2hrs. Next  embryos at desired stages were dechorionated and deyolked manually by forceps and transferred to a 1.5 ml Eppendorf tube with 50 µl DMEM/F12 media. Dissections were performed for up 15 min. The volume of DMEM/F12 meida containing embryos was adjusted to 200 µl  then cells were mechanically dissociated by flicking the tube 30 times and pipetting mixture 10 times through a 200 µl tip. The volume was adjusted to 1 ml with PBS containing 1.0% BSA  and spun to pellet cells at 300 g for 30s. The supernatant was removed and cells were resuspended in 80 µl of PBS containing 0.1% BSA and 20% Optiprep StemCell  Cat 07820  aiming for a concentration above 100 cells/µl. Cells were then passed through a cell sieve 100 µm for dome stage and 70 µm for shield stage. Single cell RNA seq library was performed with Chromium Next GEM Single Cell kit 10x Genomics  Cat PN 1000121 based on standard protocol. All libraries were sequenced by Illumina Hi Seq 1500 or 2500 or XTen platform according to manufacturer's instruction.,GEO Accession:GSM5351824,RNA-Seq,TRANSCRIPTOMIC,cDNA,PAIRED,ILLUMINA,HiSeq X Ten,,SRP322195,,loader:fastq load.py,Dome_dnmt1_mKD_scRNA_seq_rep1_1_r1.fq.gz Dome_dnmt1_mKD_scRNA_seq_rep1_1_r2.fq.gz,fastq fastq,10123733400.0,33745778.0,GSM5351824 r1,0:150 1:150,A:2581474639;C:1791650214;G:1877690939;T:3872559724;N:357884,150,150,,,2581474639,1791650214,1877690939,3872559724,357884,SRX11041469,SRS9110396,SRA1239365,GEO,"THU-PKU Center for Life Sciences, Tsinghua University",2,0.29029,0.88868,0.04884,0.06256,0.95213,0.78354,0.5511,0.50789,150,150,T,B,mate1 technical by mapping diff,illumina,hiseq_era,unknown,cdna_unspecified,unknown,sc,single_cell_droplet,10x,,China,2021-06-01,Blastula,Embryo,Embryo Imprecise,All anatomical structures
64436,SRR14703455,SRX11041469,SRS9110396,SRP322195,PRJNA734348,Methylome inheritance and enhancer dememorization construct an epigenetic gate safeguarding embryonic programs,GSE175951,Other,Unlike that of mammals  the total DNA methylome of many cold blooded vertebrates is globally inherited from gametes to early embryos. In zebrafish  this is however accompanied by sweeping “dememorization” of enhancers prior to fertilization for sperm and just post fertilization for oocyte  as they undergo full methylation and are not demethylated again until phylotypic stage. The significance of both global methylome inheritance and enhancer dememorization in early embryos remains largely unknown. Adding to the puzzles  the zygotic mutant zebrafish of dnmt1  the major DNA methylation maintenance methyltransferase  surprisingly can develop to term. To solve the role of DNA methylation in early development  we generated zebrafish embryos derived from dnmt1 knocking down oocytes using a recently developed method OMIS microinjection in situ  which successfully eliminated DNA methylation before zygotic genome activation. dnmt1 deficient embryos failed to initiate epiboly and died around gastrulation. This is in part caused by activation of immune response and p53 regulated apoptosis  likely triggered by the derepression of transposable elements. Single cell RNA seq further revealed defective differentiation in these mutants. DNA methylation is also required for the establishment of repressive histone marks H3K27me3 and H2AK119ub. Strikingly  the loss of DNA methylation leads to extensive derepression of somatic genes and enhancers  which acquire ectopic H3K27ac  accessible chromatin  and H3K4me3. These somatic enhancers are preferentially CG rich and are bound by CG containing TFs. By contrast  embryonic enhancers are generally CG poor  methylation insensitive  and are bound by CG less TFs. Hence  the global DNA methylome inheritance is essential for vertebrate early development  and enhancer dememorization resets an epigenetic gate that separates embryonic and somatic programs. Overall design: By employing MethylC seq  total RNA seq  scRNA seq  CUT&RUN  ChIP seq  and ATAC seq in control and dnmt1 mKD embryos at oocyte  256 cell  dome and shield stages in zebrafish  we interrogated the function of DNA methylome and its inheritance in early zebrafish development.,,pubmed:34936444,,Dome dnmt1 mKD scRNA seq rep1,GSM5351824,,source name:embryo|strain:AB|genotype:WT|developmental stage:Dome|tissue:embryo|treatment:microinjection with Rhodamine B and dnmt1 MO at oogenesis III stage GV,Dome dnmt1 mKD scRNA seq rep1,All STEM seq datasets were mapped to the danRer7 reference genome by Bismark Krueger and Andrews  2011. Reads were trimmed with cutadaptor Martin  2011 using parameters:   minimum length 20   pair filter=any. Alignments were performed with the following parameters:  N 1  X 600   score min L 0  0.6. Multi mapped reads and PCR duplicates were removed. bismark methylation extractor was used to calculate the DNA methylation level. Total RNA seq data were firstly processed using Trim Galore! with default parameters to trim the adapter containing and low quality reads. The filtered data were then mapped to the zebrafish reference genome danRer7 by STAR version: STAR 2.5.3a modified Dobin et al.  2013 with the following parameters:   outSAMstrandField intronMotif   outSAMattributes All   outSAMunmapped Within   outSAMattrIHstart 0   outWigStrand Stranded   outFilterMultimapNmax 20   twopassMode Basic. The gene expression level was normalized to fragments per kilobase of transcript per million mapped FPKM values using Cufflinks version 2.2.1 Trapnell et al.  2012. The scRNA seq data were processed with Cell Ranger 3.1 for genome alignment danRer10  transcript counting  and gene cell barcode matrix generation. Before the downstream analysis  cells with unique detected genes lower than <2 000 and higher than 6 000 genes were discarded  and cells with high percentage of mitochondrial genes >5% were removed too. Subtypes of cells were identified with Seurat 3.0 through the integrating of control and mKD embryos at dome and shield stages with setting control embryos as reference. To make the data comparable samples  we performed SCTransform normalization separately for each dataset. To further reduce the bais caused by sequencing depth of scRNA seq and percentage of mitochondrial genes  UMI variance and percent.mt were regressed out from SCTransform normalized data matrix by linear model with negative binomial distribution. All ChIP seq reads were aligned to the zebrafish reference genome danRer7 using Bowtie2 version 2.2.2 Langmead and Salzberg  2012 with the parameters –t –q –N 1 –L 25. All unmapped reads  non uniquely mapped reads and PCR duplicates were removed. For downstream analysis  the read counts were normalized by computing the numbers of reads per kilobase of bin per million of reads sequenced RPKM. To visualize the STAR ChIP seq signal in the UCSC genome browser  each read was extended by 250 bp and the coverage for each base was counted. Replicates of STAR ChIP seq were pooled for the downstream analysis. The ATAC seq data were mapped to the zebrafish reference genome danRer7 using Bowtie2 version 2.2.2 Langmead and Salzberg  2012 with the parameters –t –q –N 1 –L 25. All unmapped reads  non uniquely mapped reads and PCR duplicates were removed. For downstream analysis  the read counts were normalized by computing the numbers of reads per kilobase of bin per million of reads sequenced RPKM. To minimize the batch and cell type variation  RPKM values across whole genome were further Z score normalized. To visualize the CUT&RUN signals in the UCSC genome browser  each read was extended by 250 bp and the coverage for each base was counted. Genome build: danRer7 Supplementary files format and content: Matrix for scRNA seq data  gene expression table for total RNA seq data  Bigwig and narrowPeak for ChIP seq and ATAC seq data,embryo,Briefly  on the first day of OMIS Wu et al.  2018b  adult females at 5 mpf 12 mpf were anesthetized in 550 µg/ml tricaine Sigma  Cat A5040 in a petri dish. Then the fish was placed on a damp sponge with specific buffer 5.4 mM KCl  136.8 mM NaCl  4.2 mM NaHCO3  0.44 mM KH2PO4  0.25 mM Na2HPO4  and 0.5% wt/vol BSA. A cut was made on one side of belly to expose the ovary. The diluted MOs were microinjected into each oocyte. Rhodamine B Sigma  Cat R8881 was co injected with MOs as a dye. post injection  the wound on the belly was sewed with a surgical sewing needle carefully and quickly. Once the operation was done  the female was transferred into fish water supplemented with 32 µg/ml tricaine  10 unit/ml penicillin and 10 µg/ml streptomycin HyClone  Cat SV30010. Then the fish was transferred to fish water containing gradually reduced concentrations of tricaine. In the evening of the second day  the female was paired with a wild type male. In the morning of the third day  the pair started to chase and lay fertilized eggs naturally. The injected oocyte derived embryos were identified by co injected dye rhodamine B at very early developmental stages. The injection doses of dnmt1 MO were 5 ng per oocyte in the same assay. MOs were dissolved in RNase free water  and heated to 65°C for 11 min before microinjection.,Cell dissociation protocol was based on a previously described method Farrell et al.  2018 with modifications to adapt it for 10x Genomics platform. Briefly  dnmt1 mKD and control embryos were collected 20 min post fertilization and cultured as mentioned before. Then 15 embryos at dome or shield stages for each sample were transferred into plastic Petri dishes that had previously been coated with 2% agarose and soaked with DMEM/F12 media Gibco/Life Technologies  Cat 11330032 at least 2hrs. Next  embryos at desired stages were dechorionated and deyolked manually by forceps and transferred to a 1.5 ml Eppendorf tube with 50 µl DMEM/F12 media. Dissections were performed for up 15 min. The volume of DMEM/F12 meida containing embryos was adjusted to 200 µl  then cells were mechanically dissociated by flicking the tube 30 times and pipetting mixture 10 times through a 200 µl tip. The volume was adjusted to 1 ml with PBS containing 1.0% BSA  and spun to pellet cells at 300 g for 30s. The supernatant was removed and cells were resuspended in 80 µl of PBS containing 0.1% BSA and 20% Optiprep StemCell  Cat 07820  aiming for a concentration above 100 cells/µl. Cells were then passed through a cell sieve 100 µm for dome stage and 70 µm for shield stage. Single cell RNA seq library was performed with Chromium Next GEM Single Cell kit 10x Genomics  Cat PN 1000121 based on standard protocol. All libraries were sequenced by Illumina Hi Seq 1500 or 2500 or XTen platform according to manufacturer’s instruction.,The wild type AB strain was used in the experiments. The females post OMIS were fed with live adult brine shrimp thrice a day. All embryos were raised in Holtfreter’s solution at 28.5C and staged as described previously Kimmel et al.  1995.,strain:AB|genotype:WT|developmental stage:Dome|tissue:embryo|treatment:microinjection with Rhodamine B and dnmt1 MO at oogenesis III stage GV,GSM5351824,GSM5351824: Dome dnmt1 mKD scRNA seq rep1; Danio rerio; RNA Seq,GSM5351824,,1,Cell dissociation protocol was based on a previously described method Farrell et al.  2018 with modifications to adapt it for 10x Genomics platform. Briefly  dnmt1 mKD and control embryos were collected 20 min post fertilization and cultured as mentioned before. Then 15 embryos at dome or shield stages for each sample were transferred into plastic Petri dishes that had previously been coated with 2% agarose and soaked with DMEM/F12 media Gibco/Life Technologies  Cat 11330032 at least 2hrs. Next  embryos at desired stages were dechorionated and deyolked manually by forceps and transferred to a 1.5 ml Eppendorf tube with 50 µl DMEM/F12 media. Dissections were performed for up 15 min. The volume of DMEM/F12 meida containing embryos was adjusted to 200 µl  then cells were mechanically dissociated by flicking the tube 30 times and pipetting mixture 10 times through a 200 µl tip. The volume was adjusted to 1 ml with PBS containing 1.0% BSA  and spun to pellet cells at 300 g for 30s. The supernatant was removed and cells were resuspended in 80 µl of PBS containing 0.1% BSA and 20% Optiprep StemCell  Cat 07820  aiming for a concentration above 100 cells/µl. Cells were then passed through a cell sieve 100 µm for dome stage and 70 µm for shield stage. Single cell RNA seq library was performed with Chromium Next GEM Single Cell kit 10x Genomics  Cat PN 1000121 based on standard protocol. All libraries were sequenced by Illumina Hi Seq 1500 or 2500 or XTen platform according to manufacturer's instruction.,GEO Accession:GSM5351824,RNA-Seq,TRANSCRIPTOMIC,cDNA,PAIRED,ILLUMINA,HiSeq X Ten,,SRP322195,,loader:fastq load.py,Dome_dnmt1_mKD_scRNA_seq_rep1_2_r1.fq.gz Dome_dnmt1_mKD_scRNA_seq_rep1_2_r2.fq.gz,fastq fastq,10994964300.0,36649881.0,GSM5351824 r2,0:150 1:150,A:2803461151;C:1955085347;G:2043816454;T:4192215113;N:386235,150,150,,,2803461151,1955085347,2043816454,4192215113,386235,SRX11041469,SRS9110396,SRA1239365,GEO,"THU-PKU Center for Life Sciences, Tsinghua University",2,0.29959,0.88808,0.05118,0.0642,0.95306,0.78585,0.56812,0.51636,150,150,B,B,mate1-mate2 similar by mapping diff,illumina,hiseq_era,unknown,cdna_unspecified,unknown,sc,single_cell_droplet,10x,,China,2021-06-01,Blastula,Embryo,Embryo Imprecise,All anatomical structures
64437,SRR14703456,SRX11041469,SRS9110396,SRP322195,PRJNA734348,Methylome inheritance and enhancer dememorization construct an epigenetic gate safeguarding embryonic programs,GSE175951,Other,Unlike that of mammals  the total DNA methylome of many cold blooded vertebrates is globally inherited from gametes to early embryos. In zebrafish  this is however accompanied by sweeping “dememorization” of enhancers prior to fertilization for sperm and just post fertilization for oocyte  as they undergo full methylation and are not demethylated again until phylotypic stage. The significance of both global methylome inheritance and enhancer dememorization in early embryos remains largely unknown. Adding to the puzzles  the zygotic mutant zebrafish of dnmt1  the major DNA methylation maintenance methyltransferase  surprisingly can develop to term. To solve the role of DNA methylation in early development  we generated zebrafish embryos derived from dnmt1 knocking down oocytes using a recently developed method OMIS microinjection in situ  which successfully eliminated DNA methylation before zygotic genome activation. dnmt1 deficient embryos failed to initiate epiboly and died around gastrulation. This is in part caused by activation of immune response and p53 regulated apoptosis  likely triggered by the derepression of transposable elements. Single cell RNA seq further revealed defective differentiation in these mutants. DNA methylation is also required for the establishment of repressive histone marks H3K27me3 and H2AK119ub. Strikingly  the loss of DNA methylation leads to extensive derepression of somatic genes and enhancers  which acquire ectopic H3K27ac  accessible chromatin  and H3K4me3. These somatic enhancers are preferentially CG rich and are bound by CG containing TFs. By contrast  embryonic enhancers are generally CG poor  methylation insensitive  and are bound by CG less TFs. Hence  the global DNA methylome inheritance is essential for vertebrate early development  and enhancer dememorization resets an epigenetic gate that separates embryonic and somatic programs. Overall design: By employing MethylC seq  total RNA seq  scRNA seq  CUT&RUN  ChIP seq  and ATAC seq in control and dnmt1 mKD embryos at oocyte  256 cell  dome and shield stages in zebrafish  we interrogated the function of DNA methylome and its inheritance in early zebrafish development.,,pubmed:34936444,,Dome dnmt1 mKD scRNA seq rep1,GSM5351824,,source name:embryo|strain:AB|genotype:WT|developmental stage:Dome|tissue:embryo|treatment:microinjection with Rhodamine B and dnmt1 MO at oogenesis III stage GV,Dome dnmt1 mKD scRNA seq rep1,All STEM seq datasets were mapped to the danRer7 reference genome by Bismark Krueger and Andrews  2011. Reads were trimmed with cutadaptor Martin  2011 using parameters:   minimum length 20   pair filter=any. Alignments were performed with the following parameters:  N 1  X 600   score min L 0  0.6. Multi mapped reads and PCR duplicates were removed. bismark methylation extractor was used to calculate the DNA methylation level. Total RNA seq data were firstly processed using Trim Galore! with default parameters to trim the adapter containing and low quality reads. The filtered data were then mapped to the zebrafish reference genome danRer7 by STAR version: STAR 2.5.3a modified Dobin et al.  2013 with the following parameters:   outSAMstrandField intronMotif   outSAMattributes All   outSAMunmapped Within   outSAMattrIHstart 0   outWigStrand Stranded   outFilterMultimapNmax 20   twopassMode Basic. The gene expression level was normalized to fragments per kilobase of transcript per million mapped FPKM values using Cufflinks version 2.2.1 Trapnell et al.  2012. The scRNA seq data were processed with Cell Ranger 3.1 for genome alignment danRer10  transcript counting  and gene cell barcode matrix generation. Before the downstream analysis  cells with unique detected genes lower than <2 000 and higher than 6 000 genes were discarded  and cells with high percentage of mitochondrial genes >5% were removed too. Subtypes of cells were identified with Seurat 3.0 through the integrating of control and mKD embryos at dome and shield stages with setting control embryos as reference. To make the data comparable samples  we performed SCTransform normalization separately for each dataset. To further reduce the bais caused by sequencing depth of scRNA seq and percentage of mitochondrial genes  UMI variance and percent.mt were regressed out from SCTransform normalized data matrix by linear model with negative binomial distribution. All ChIP seq reads were aligned to the zebrafish reference genome danRer7 using Bowtie2 version 2.2.2 Langmead and Salzberg  2012 with the parameters –t –q –N 1 –L 25. All unmapped reads  non uniquely mapped reads and PCR duplicates were removed. For downstream analysis  the read counts were normalized by computing the numbers of reads per kilobase of bin per million of reads sequenced RPKM. To visualize the STAR ChIP seq signal in the UCSC genome browser  each read was extended by 250 bp and the coverage for each base was counted. Replicates of STAR ChIP seq were pooled for the downstream analysis. The ATAC seq data were mapped to the zebrafish reference genome danRer7 using Bowtie2 version 2.2.2 Langmead and Salzberg  2012 with the parameters –t –q –N 1 –L 25. All unmapped reads  non uniquely mapped reads and PCR duplicates were removed. For downstream analysis  the read counts were normalized by computing the numbers of reads per kilobase of bin per million of reads sequenced RPKM. To minimize the batch and cell type variation  RPKM values across whole genome were further Z score normalized. To visualize the CUT&RUN signals in the UCSC genome browser  each read was extended by 250 bp and the coverage for each base was counted. Genome build: danRer7 Supplementary files format and content: Matrix for scRNA seq data  gene expression table for total RNA seq data  Bigwig and narrowPeak for ChIP seq and ATAC seq data,embryo,Briefly  on the first day of OMIS Wu et al.  2018b  adult females at 5 mpf 12 mpf were anesthetized in 550 µg/ml tricaine Sigma  Cat A5040 in a petri dish. Then the fish was placed on a damp sponge with specific buffer 5.4 mM KCl  136.8 mM NaCl  4.2 mM NaHCO3  0.44 mM KH2PO4  0.25 mM Na2HPO4  and 0.5% wt/vol BSA. A cut was made on one side of belly to expose the ovary. The diluted MOs were microinjected into each oocyte. Rhodamine B Sigma  Cat R8881 was co injected with MOs as a dye. post injection  the wound on the belly was sewed with a surgical sewing needle carefully and quickly. Once the operation was done  the female was transferred into fish water supplemented with 32 µg/ml tricaine  10 unit/ml penicillin and 10 µg/ml streptomycin HyClone  Cat SV30010. Then the fish was transferred to fish water containing gradually reduced concentrations of tricaine. In the evening of the second day  the female was paired with a wild type male. In the morning of the third day  the pair started to chase and lay fertilized eggs naturally. The injected oocyte derived embryos were identified by co injected dye rhodamine B at very early developmental stages. The injection doses of dnmt1 MO were 5 ng per oocyte in the same assay. MOs were dissolved in RNase free water  and heated to 65°C for 11 min before microinjection.,Cell dissociation protocol was based on a previously described method Farrell et al.  2018 with modifications to adapt it for 10x Genomics platform. Briefly  dnmt1 mKD and control embryos were collected 20 min post fertilization and cultured as mentioned before. Then 15 embryos at dome or shield stages for each sample were transferred into plastic Petri dishes that had previously been coated with 2% agarose and soaked with DMEM/F12 media Gibco/Life Technologies  Cat 11330032 at least 2hrs. Next  embryos at desired stages were dechorionated and deyolked manually by forceps and transferred to a 1.5 ml Eppendorf tube with 50 µl DMEM/F12 media. Dissections were performed for up 15 min. The volume of DMEM/F12 meida containing embryos was adjusted to 200 µl  then cells were mechanically dissociated by flicking the tube 30 times and pipetting mixture 10 times through a 200 µl tip. The volume was adjusted to 1 ml with PBS containing 1.0% BSA  and spun to pellet cells at 300 g for 30s. The supernatant was removed and cells were resuspended in 80 µl of PBS containing 0.1% BSA and 20% Optiprep StemCell  Cat 07820  aiming for a concentration above 100 cells/µl. Cells were then passed through a cell sieve 100 µm for dome stage and 70 µm for shield stage. Single cell RNA seq library was performed with Chromium Next GEM Single Cell kit 10x Genomics  Cat PN 1000121 based on standard protocol. All libraries were sequenced by Illumina Hi Seq 1500 or 2500 or XTen platform according to manufacturer’s instruction.,The wild type AB strain was used in the experiments. The females post OMIS were fed with live adult brine shrimp thrice a day. All embryos were raised in Holtfreter’s solution at 28.5C and staged as described previously Kimmel et al.  1995.,strain:AB|genotype:WT|developmental stage:Dome|tissue:embryo|treatment:microinjection with Rhodamine B and dnmt1 MO at oogenesis III stage GV,GSM5351824,GSM5351824: Dome dnmt1 mKD scRNA seq rep1; Danio rerio; RNA Seq,GSM5351824,,1,Cell dissociation protocol was based on a previously described method Farrell et al.  2018 with modifications to adapt it for 10x Genomics platform. Briefly  dnmt1 mKD and control embryos were collected 20 min post fertilization and cultured as mentioned before. Then 15 embryos at dome or shield stages for each sample were transferred into plastic Petri dishes that had previously been coated with 2% agarose and soaked with DMEM/F12 media Gibco/Life Technologies  Cat 11330032 at least 2hrs. Next  embryos at desired stages were dechorionated and deyolked manually by forceps and transferred to a 1.5 ml Eppendorf tube with 50 µl DMEM/F12 media. Dissections were performed for up 15 min. The volume of DMEM/F12 meida containing embryos was adjusted to 200 µl  then cells were mechanically dissociated by flicking the tube 30 times and pipetting mixture 10 times through a 200 µl tip. The volume was adjusted to 1 ml with PBS containing 1.0% BSA  and spun to pellet cells at 300 g for 30s. The supernatant was removed and cells were resuspended in 80 µl of PBS containing 0.1% BSA and 20% Optiprep StemCell  Cat 07820  aiming for a concentration above 100 cells/µl. Cells were then passed through a cell sieve 100 µm for dome stage and 70 µm for shield stage. Single cell RNA seq library was performed with Chromium Next GEM Single Cell kit 10x Genomics  Cat PN 1000121 based on standard protocol. All libraries were sequenced by Illumina Hi Seq 1500 or 2500 or XTen platform according to manufacturer's instruction.,GEO Accession:GSM5351824,RNA-Seq,TRANSCRIPTOMIC,cDNA,PAIRED,ILLUMINA,HiSeq X Ten,,SRP322195,,loader:fastq load.py,Dome_dnmt1_mKD_scRNA_seq_rep1_3_r1.fq.gz Dome_dnmt1_mKD_scRNA_seq_rep1_3_r2.fq.gz,fastq fastq,10593519300.0,35311731.0,GSM5351824 r3,0:150 1:150,A:2701228305;C:1877396474;G:1968127865;T:4046392949;N:373707,150,150,,,2701228305,1877396474,1968127865,4046392949,373707,SRX11041469,SRS9110396,SRA1239365,GEO,"THU-PKU Center for Life Sciences, Tsinghua University",2,0.30137,0.88928,0.05094,0.0651,0.95237,0.78468,0.57223,0.51184,150,150,B,B,mate1-mate2 similar by mapping diff,illumina,hiseq_era,unknown,cdna_unspecified,unknown,sc,single_cell_droplet,10x,,China,2021-06-01,Blastula,Embryo,Embryo Imprecise,All anatomical structures
64438,SRR14703457,SRX11041469,SRS9110396,SRP322195,PRJNA734348,Methylome inheritance and enhancer dememorization construct an epigenetic gate safeguarding embryonic programs,GSE175951,Other,Unlike that of mammals  the total DNA methylome of many cold blooded vertebrates is globally inherited from gametes to early embryos. In zebrafish  this is however accompanied by sweeping “dememorization” of enhancers prior to fertilization for sperm and just post fertilization for oocyte  as they undergo full methylation and are not demethylated again until phylotypic stage. The significance of both global methylome inheritance and enhancer dememorization in early embryos remains largely unknown. Adding to the puzzles  the zygotic mutant zebrafish of dnmt1  the major DNA methylation maintenance methyltransferase  surprisingly can develop to term. To solve the role of DNA methylation in early development  we generated zebrafish embryos derived from dnmt1 knocking down oocytes using a recently developed method OMIS microinjection in situ  which successfully eliminated DNA methylation before zygotic genome activation. dnmt1 deficient embryos failed to initiate epiboly and died around gastrulation. This is in part caused by activation of immune response and p53 regulated apoptosis  likely triggered by the derepression of transposable elements. Single cell RNA seq further revealed defective differentiation in these mutants. DNA methylation is also required for the establishment of repressive histone marks H3K27me3 and H2AK119ub. Strikingly  the loss of DNA methylation leads to extensive derepression of somatic genes and enhancers  which acquire ectopic H3K27ac  accessible chromatin  and H3K4me3. These somatic enhancers are preferentially CG rich and are bound by CG containing TFs. By contrast  embryonic enhancers are generally CG poor  methylation insensitive  and are bound by CG less TFs. Hence  the global DNA methylome inheritance is essential for vertebrate early development  and enhancer dememorization resets an epigenetic gate that separates embryonic and somatic programs. Overall design: By employing MethylC seq  total RNA seq  scRNA seq  CUT&RUN  ChIP seq  and ATAC seq in control and dnmt1 mKD embryos at oocyte  256 cell  dome and shield stages in zebrafish  we interrogated the function of DNA methylome and its inheritance in early zebrafish development.,,pubmed:34936444,,Dome dnmt1 mKD scRNA seq rep1,GSM5351824,,source name:embryo|strain:AB|genotype:WT|developmental stage:Dome|tissue:embryo|treatment:microinjection with Rhodamine B and dnmt1 MO at oogenesis III stage GV,Dome dnmt1 mKD scRNA seq rep1,All STEM seq datasets were mapped to the danRer7 reference genome by Bismark Krueger and Andrews  2011. Reads were trimmed with cutadaptor Martin  2011 using parameters:   minimum length 20   pair filter=any. Alignments were performed with the following parameters:  N 1  X 600   score min L 0  0.6. Multi mapped reads and PCR duplicates were removed. bismark methylation extractor was used to calculate the DNA methylation level. Total RNA seq data were firstly processed using Trim Galore! with default parameters to trim the adapter containing and low quality reads. The filtered data were then mapped to the zebrafish reference genome danRer7 by STAR version: STAR 2.5.3a modified Dobin et al.  2013 with the following parameters:   outSAMstrandField intronMotif   outSAMattributes All   outSAMunmapped Within   outSAMattrIHstart 0   outWigStrand Stranded   outFilterMultimapNmax 20   twopassMode Basic. The gene expression level was normalized to fragments per kilobase of transcript per million mapped FPKM values using Cufflinks version 2.2.1 Trapnell et al.  2012. The scRNA seq data were processed with Cell Ranger 3.1 for genome alignment danRer10  transcript counting  and gene cell barcode matrix generation. Before the downstream analysis  cells with unique detected genes lower than <2 000 and higher than 6 000 genes were discarded  and cells with high percentage of mitochondrial genes >5% were removed too. Subtypes of cells were identified with Seurat 3.0 through the integrating of control and mKD embryos at dome and shield stages with setting control embryos as reference. To make the data comparable samples  we performed SCTransform normalization separately for each dataset. To further reduce the bais caused by sequencing depth of scRNA seq and percentage of mitochondrial genes  UMI variance and percent.mt were regressed out from SCTransform normalized data matrix by linear model with negative binomial distribution. All ChIP seq reads were aligned to the zebrafish reference genome danRer7 using Bowtie2 version 2.2.2 Langmead and Salzberg  2012 with the parameters –t –q –N 1 –L 25. All unmapped reads  non uniquely mapped reads and PCR duplicates were removed. For downstream analysis  the read counts were normalized by computing the numbers of reads per kilobase of bin per million of reads sequenced RPKM. To visualize the STAR ChIP seq signal in the UCSC genome browser  each read was extended by 250 bp and the coverage for each base was counted. Replicates of STAR ChIP seq were pooled for the downstream analysis. The ATAC seq data were mapped to the zebrafish reference genome danRer7 using Bowtie2 version 2.2.2 Langmead and Salzberg  2012 with the parameters –t –q –N 1 –L 25. All unmapped reads  non uniquely mapped reads and PCR duplicates were removed. For downstream analysis  the read counts were normalized by computing the numbers of reads per kilobase of bin per million of reads sequenced RPKM. To minimize the batch and cell type variation  RPKM values across whole genome were further Z score normalized. To visualize the CUT&RUN signals in the UCSC genome browser  each read was extended by 250 bp and the coverage for each base was counted. Genome build: danRer7 Supplementary files format and content: Matrix for scRNA seq data  gene expression table for total RNA seq data  Bigwig and narrowPeak for ChIP seq and ATAC seq data,embryo,Briefly  on the first day of OMIS Wu et al.  2018b  adult females at 5 mpf 12 mpf were anesthetized in 550 µg/ml tricaine Sigma  Cat A5040 in a petri dish. Then the fish was placed on a damp sponge with specific buffer 5.4 mM KCl  136.8 mM NaCl  4.2 mM NaHCO3  0.44 mM KH2PO4  0.25 mM Na2HPO4  and 0.5% wt/vol BSA. A cut was made on one side of belly to expose the ovary. The diluted MOs were microinjected into each oocyte. Rhodamine B Sigma  Cat R8881 was co injected with MOs as a dye. post injection  the wound on the belly was sewed with a surgical sewing needle carefully and quickly. Once the operation was done  the female was transferred into fish water supplemented with 32 µg/ml tricaine  10 unit/ml penicillin and 10 µg/ml streptomycin HyClone  Cat SV30010. Then the fish was transferred to fish water containing gradually reduced concentrations of tricaine. In the evening of the second day  the female was paired with a wild type male. In the morning of the third day  the pair started to chase and lay fertilized eggs naturally. The injected oocyte derived embryos were identified by co injected dye rhodamine B at very early developmental stages. The injection doses of dnmt1 MO were 5 ng per oocyte in the same assay. MOs were dissolved in RNase free water  and heated to 65°C for 11 min before microinjection.,Cell dissociation protocol was based on a previously described method Farrell et al.  2018 with modifications to adapt it for 10x Genomics platform. Briefly  dnmt1 mKD and control embryos were collected 20 min post fertilization and cultured as mentioned before. Then 15 embryos at dome or shield stages for each sample were transferred into plastic Petri dishes that had previously been coated with 2% agarose and soaked with DMEM/F12 media Gibco/Life Technologies  Cat 11330032 at least 2hrs. Next  embryos at desired stages were dechorionated and deyolked manually by forceps and transferred to a 1.5 ml Eppendorf tube with 50 µl DMEM/F12 media. Dissections were performed for up 15 min. The volume of DMEM/F12 meida containing embryos was adjusted to 200 µl  then cells were mechanically dissociated by flicking the tube 30 times and pipetting mixture 10 times through a 200 µl tip. The volume was adjusted to 1 ml with PBS containing 1.0% BSA  and spun to pellet cells at 300 g for 30s. The supernatant was removed and cells were resuspended in 80 µl of PBS containing 0.1% BSA and 20% Optiprep StemCell  Cat 07820  aiming for a concentration above 100 cells/µl. Cells were then passed through a cell sieve 100 µm for dome stage and 70 µm for shield stage. Single cell RNA seq library was performed with Chromium Next GEM Single Cell kit 10x Genomics  Cat PN 1000121 based on standard protocol. All libraries were sequenced by Illumina Hi Seq 1500 or 2500 or XTen platform according to manufacturer’s instruction.,The wild type AB strain was used in the experiments. The females post OMIS were fed with live adult brine shrimp thrice a day. All embryos were raised in Holtfreter’s solution at 28.5C and staged as described previously Kimmel et al.  1995.,strain:AB|genotype:WT|developmental stage:Dome|tissue:embryo|treatment:microinjection with Rhodamine B and dnmt1 MO at oogenesis III stage GV,GSM5351824,GSM5351824: Dome dnmt1 mKD scRNA seq rep1; Danio rerio; RNA Seq,GSM5351824,,1,Cell dissociation protocol was based on a previously described method Farrell et al.  2018 with modifications to adapt it for 10x Genomics platform. Briefly  dnmt1 mKD and control embryos were collected 20 min post fertilization and cultured as mentioned before. Then 15 embryos at dome or shield stages for each sample were transferred into plastic Petri dishes that had previously been coated with 2% agarose and soaked with DMEM/F12 media Gibco/Life Technologies  Cat 11330032 at least 2hrs. Next  embryos at desired stages were dechorionated and deyolked manually by forceps and transferred to a 1.5 ml Eppendorf tube with 50 µl DMEM/F12 media. Dissections were performed for up 15 min. The volume of DMEM/F12 meida containing embryos was adjusted to 200 µl  then cells were mechanically dissociated by flicking the tube 30 times and pipetting mixture 10 times through a 200 µl tip. The volume was adjusted to 1 ml with PBS containing 1.0% BSA  and spun to pellet cells at 300 g for 30s. The supernatant was removed and cells were resuspended in 80 µl of PBS containing 0.1% BSA and 20% Optiprep StemCell  Cat 07820  aiming for a concentration above 100 cells/µl. Cells were then passed through a cell sieve 100 µm for dome stage and 70 µm for shield stage. Single cell RNA seq library was performed with Chromium Next GEM Single Cell kit 10x Genomics  Cat PN 1000121 based on standard protocol. All libraries were sequenced by Illumina Hi Seq 1500 or 2500 or XTen platform according to manufacturer's instruction.,GEO Accession:GSM5351824,RNA-Seq,TRANSCRIPTOMIC,cDNA,PAIRED,ILLUMINA,HiSeq X Ten,,SRP322195,,loader:fastq load.py,Dome_dnmt1_mKD_scRNA_seq_rep1_4_r1.fq.gz Dome_dnmt1_mKD_scRNA_seq_rep1_4_r2.fq.gz,fastq fastq,11575953600.0,38586512.0,GSM5351824 r4,0:150 1:150,A:2946049698;C:2060828022;G:2161335727;T:4407334347;N:405806,150,150,,,2946049698,2060828022,2161335727,4407334347,405806,SRX11041469,SRS9110396,SRA1239365,GEO,"THU-PKU Center for Life Sciences, Tsinghua University",2,0.2964,0.8886,0.05121,0.06386,0.95315,0.78577,0.54196,0.51143,150,150,T,B,mate1 technical by mapping diff,illumina,hiseq_era,unknown,cdna_unspecified,unknown,sc,single_cell_droplet,10x,,China,2021-06-01,Blastula,Embryo,Embryo Imprecise,All anatomical structures
64439,SRR14703450,SRX11041468,SRS9110395,SRP322195,PRJNA734348,Methylome inheritance and enhancer dememorization construct an epigenetic gate safeguarding embryonic programs,GSE175951,Other,Unlike that of mammals  the total DNA methylome of many cold blooded vertebrates is globally inherited from gametes to early embryos. In zebrafish  this is however accompanied by sweeping “dememorization” of enhancers prior to fertilization for sperm and just post fertilization for oocyte  as they undergo full methylation and are not demethylated again until phylotypic stage. The significance of both global methylome inheritance and enhancer dememorization in early embryos remains largely unknown. Adding to the puzzles  the zygotic mutant zebrafish of dnmt1  the major DNA methylation maintenance methyltransferase  surprisingly can develop to term. To solve the role of DNA methylation in early development  we generated zebrafish embryos derived from dnmt1 knocking down oocytes using a recently developed method OMIS microinjection in situ  which successfully eliminated DNA methylation before zygotic genome activation. dnmt1 deficient embryos failed to initiate epiboly and died around gastrulation. This is in part caused by activation of immune response and p53 regulated apoptosis  likely triggered by the derepression of transposable elements. Single cell RNA seq further revealed defective differentiation in these mutants. DNA methylation is also required for the establishment of repressive histone marks H3K27me3 and H2AK119ub. Strikingly  the loss of DNA methylation leads to extensive derepression of somatic genes and enhancers  which acquire ectopic H3K27ac  accessible chromatin  and H3K4me3. These somatic enhancers are preferentially CG rich and are bound by CG containing TFs. By contrast  embryonic enhancers are generally CG poor  methylation insensitive  and are bound by CG less TFs. Hence  the global DNA methylome inheritance is essential for vertebrate early development  and enhancer dememorization resets an epigenetic gate that separates embryonic and somatic programs. Overall design: By employing MethylC seq  total RNA seq  scRNA seq  CUT&RUN  ChIP seq  and ATAC seq in control and dnmt1 mKD embryos at oocyte  256 cell  dome and shield stages in zebrafish  we interrogated the function of DNA methylome and its inheritance in early zebrafish development.,,pubmed:34936444,,Dome ctrl scRNA seq rep2,GSM5351823,,source name:embryo|strain:AB|genotype:WT|developmental stage:Dome|tissue:embryo|treatment:microinjection with Rhodamine B and control MO at oogenesis III stage GV,Dome ctrl scRNA seq rep2,All STEM seq datasets were mapped to the danRer7 reference genome by Bismark Krueger and Andrews  2011. Reads were trimmed with cutadaptor Martin  2011 using parameters:   minimum length 20   pair filter=any. Alignments were performed with the following parameters:  N 1  X 600   score min L 0  0.6. Multi mapped reads and PCR duplicates were removed. bismark methylation extractor was used to calculate the DNA methylation level. Total RNA seq data were firstly processed using Trim Galore! with default parameters to trim the adapter containing and low quality reads. The filtered data were then mapped to the zebrafish reference genome danRer7 by STAR version: STAR 2.5.3a modified Dobin et al.  2013 with the following parameters:   outSAMstrandField intronMotif   outSAMattributes All   outSAMunmapped Within   outSAMattrIHstart 0   outWigStrand Stranded   outFilterMultimapNmax 20   twopassMode Basic. The gene expression level was normalized to fragments per kilobase of transcript per million mapped FPKM values using Cufflinks version 2.2.1 Trapnell et al.  2012. The scRNA seq data were processed with Cell Ranger 3.1 for genome alignment danRer10  transcript counting  and gene cell barcode matrix generation. Before the downstream analysis  cells with unique detected genes lower than <2 000 and higher than 6 000 genes were discarded  and cells with high percentage of mitochondrial genes >5% were removed too. Subtypes of cells were identified with Seurat 3.0 through the integrating of control and mKD embryos at dome and shield stages with setting control embryos as reference. To make the data comparable samples  we performed SCTransform normalization separately for each dataset. To further reduce the bais caused by sequencing depth of scRNA seq and percentage of mitochondrial genes  UMI variance and percent.mt were regressed out from SCTransform normalized data matrix by linear model with negative binomial distribution. All ChIP seq reads were aligned to the zebrafish reference genome danRer7 using Bowtie2 version 2.2.2 Langmead and Salzberg  2012 with the parameters –t –q –N 1 –L 25. All unmapped reads  non uniquely mapped reads and PCR duplicates were removed. For downstream analysis  the read counts were normalized by computing the numbers of reads per kilobase of bin per million of reads sequenced RPKM. To visualize the STAR ChIP seq signal in the UCSC genome browser  each read was extended by 250 bp and the coverage for each base was counted. Replicates of STAR ChIP seq were pooled for the downstream analysis. The ATAC seq data were mapped to the zebrafish reference genome danRer7 using Bowtie2 version 2.2.2 Langmead and Salzberg  2012 with the parameters –t –q –N 1 –L 25. All unmapped reads  non uniquely mapped reads and PCR duplicates were removed. For downstream analysis  the read counts were normalized by computing the numbers of reads per kilobase of bin per million of reads sequenced RPKM. To minimize the batch and cell type variation  RPKM values across whole genome were further Z score normalized. To visualize the CUT&RUN signals in the UCSC genome browser  each read was extended by 250 bp and the coverage for each base was counted. Genome build: danRer7 Supplementary files format and content: Matrix for scRNA seq data  gene expression table for total RNA seq data  Bigwig and narrowPeak for ChIP seq and ATAC seq data,embryo,Briefly  on the first day of OMIS Wu et al.  2018b  adult females at 5 mpf 12 mpf were anesthetized in 550 µg/ml tricaine Sigma  Cat A5040 in a petri dish. Then the fish was placed on a damp sponge with specific buffer 5.4 mM KCl  136.8 mM NaCl  4.2 mM NaHCO3  0.44 mM KH2PO4  0.25 mM Na2HPO4  and 0.5% wt/vol BSA. A cut was made on one side of belly to expose the ovary. The diluted MOs were microinjected into each oocyte. Rhodamine B Sigma  Cat R8881 was co injected with MOs as a dye. post injection  the wound on the belly was sewed with a surgical sewing needle carefully and quickly. Once the operation was done  the female was transferred into fish water supplemented with 32 µg/ml tricaine  10 unit/ml penicillin and 10 µg/ml streptomycin HyClone  Cat SV30010. Then the fish was transferred to fish water containing gradually reduced concentrations of tricaine. In the evening of the second day  the female was paired with a wild type male. In the morning of the third day  the pair started to chase and lay fertilized eggs naturally. The injected oocyte derived embryos were identified by co injected dye rhodamine B at very early developmental stages. The injection doses of standard control MO cMO were 5 ng per oocyte in the same assay. MOs were dissolved in RNase free water  and heated to 65°C for 10 min before microinjection.,Cell dissociation protocol was based on a previously described method Farrell et al.  2018 with modifications to adapt it for 10x Genomics platform. Briefly  dnmt1 mKD and control embryos were collected 20 min post fertilization and cultured as mentioned before. Then 15 embryos at dome or shield stages for each sample were transferred into plastic Petri dishes that had previously been coated with 2% agarose and soaked with DMEM/F12 media Gibco/Life Technologies  Cat 11330032 at least 2hrs. Next  embryos at desired stages were dechorionated and deyolked manually by forceps and transferred to a 1.5 ml Eppendorf tube with 50 µl DMEM/F12 media. Dissections were performed for up 15 min. The volume of DMEM/F12 meida containing embryos was adjusted to 200 µl  then cells were mechanically dissociated by flicking the tube 30 times and pipetting mixture 10 times through a 200 µl tip. The volume was adjusted to 1 ml with PBS containing 1.0% BSA  and spun to pellet cells at 300 g for 30s. The supernatant was removed and cells were resuspended in 80 µl of PBS containing 0.1% BSA and 20% Optiprep StemCell  Cat 07820  aiming for a concentration above 100 cells/µl. Cells were then passed through a cell sieve 100 µm for dome stage and 70 µm for shield stage. Single cell RNA seq library was performed with Chromium Next GEM Single Cell kit 10x Genomics  Cat PN 1000121 based on standard protocol. All libraries were sequenced by Illumina Hi Seq 1500 or 2500 or XTen platform according to manufacturer’s instruction.,The wild type AB strain was used in the experiments. The females post OMIS were fed with live adult brine shrimp thrice a day. All embryos were raised in Holtfreter’s solution at 28.5C and staged as described previously Kimmel et al.  1995.,strain:AB|genotype:WT|developmental stage:Dome|tissue:embryo|treatment:microinjection with Rhodamine B and control MO at oogenesis III stage GV,GSM5351823,GSM5351823: Dome ctrl scRNA seq rep2; Danio rerio; RNA Seq,GSM5351823,,1,Cell dissociation protocol was based on a previously described method Farrell et al.  2018 with modifications to adapt it for 10x Genomics platform. Briefly  dnmt1 mKD and control embryos were collected 20 min post fertilization and cultured as mentioned before. Then 15 embryos at dome or shield stages for each sample were transferred into plastic Petri dishes that had previously been coated with 2% agarose and soaked with DMEM/F12 media Gibco/Life Technologies  Cat 11330032 at least 2hrs. Next  embryos at desired stages were dechorionated and deyolked manually by forceps and transferred to a 1.5 ml Eppendorf tube with 50 µl DMEM/F12 media. Dissections were performed for up 15 min. The volume of DMEM/F12 meida containing embryos was adjusted to 200 µl  then cells were mechanically dissociated by flicking the tube 30 times and pipetting mixture 10 times through a 200 µl tip. The volume was adjusted to 1 ml with PBS containing 1.0% BSA  and spun to pellet cells at 300 g for 30s. The supernatant was removed and cells were resuspended in 80 µl of PBS containing 0.1% BSA and 20% Optiprep StemCell  Cat 07820  aiming for a concentration above 100 cells/µl. Cells were then passed through a cell sieve 100 µm for dome stage and 70 µm for shield stage. Single cell RNA seq library was performed with Chromium Next GEM Single Cell kit 10x Genomics  Cat PN 1000121 based on standard protocol. All libraries were sequenced by Illumina Hi Seq 1500 or 2500 or XTen platform according to manufacturer's instruction.,GEO Accession:GSM5351823,RNA-Seq,TRANSCRIPTOMIC,cDNA,PAIRED,ILLUMINA,Illumina HiSeq 1500,,SRP322195,,loader:fastq load.py,Dome_ctrl_scRNA_seq_rep2_1_r1.fq.gz Dome_ctrl_scRNA_seq_rep2_1_r2.fq.gz,fastq fastq,4469388000.0,14897960.0,GSM5351823 r1,0:150 1:150,A:1118783090;C:835285829;G:877227752;T:1637935858;N:155471,150,150,,,1118783090,835285829,877227752,1637935858,155471,SRX11041468,SRS9110395,SRA1239365,GEO,"THU-PKU Center for Life Sciences, Tsinghua University",2,0.40164,0.87838,0.09742,0.14574,0.94501,0.79788,0.5625,0.54079,150,150,B,B,mate1-mate2 similar by mapping diff,illumina,hiseq_era,unknown,cdna_unspecified,unknown,sc,single_cell_droplet,10x,,China,2021-06-01,Blastula,Embryo,Embryo Imprecise,All anatomical structures
64440,SRR14703451,SRX11041468,SRS9110395,SRP322195,PRJNA734348,Methylome inheritance and enhancer dememorization construct an epigenetic gate safeguarding embryonic programs,GSE175951,Other,Unlike that of mammals  the total DNA methylome of many cold blooded vertebrates is globally inherited from gametes to early embryos. In zebrafish  this is however accompanied by sweeping “dememorization” of enhancers prior to fertilization for sperm and just post fertilization for oocyte  as they undergo full methylation and are not demethylated again until phylotypic stage. The significance of both global methylome inheritance and enhancer dememorization in early embryos remains largely unknown. Adding to the puzzles  the zygotic mutant zebrafish of dnmt1  the major DNA methylation maintenance methyltransferase  surprisingly can develop to term. To solve the role of DNA methylation in early development  we generated zebrafish embryos derived from dnmt1 knocking down oocytes using a recently developed method OMIS microinjection in situ  which successfully eliminated DNA methylation before zygotic genome activation. dnmt1 deficient embryos failed to initiate epiboly and died around gastrulation. This is in part caused by activation of immune response and p53 regulated apoptosis  likely triggered by the derepression of transposable elements. Single cell RNA seq further revealed defective differentiation in these mutants. DNA methylation is also required for the establishment of repressive histone marks H3K27me3 and H2AK119ub. Strikingly  the loss of DNA methylation leads to extensive derepression of somatic genes and enhancers  which acquire ectopic H3K27ac  accessible chromatin  and H3K4me3. These somatic enhancers are preferentially CG rich and are bound by CG containing TFs. By contrast  embryonic enhancers are generally CG poor  methylation insensitive  and are bound by CG less TFs. Hence  the global DNA methylome inheritance is essential for vertebrate early development  and enhancer dememorization resets an epigenetic gate that separates embryonic and somatic programs. Overall design: By employing MethylC seq  total RNA seq  scRNA seq  CUT&RUN  ChIP seq  and ATAC seq in control and dnmt1 mKD embryos at oocyte  256 cell  dome and shield stages in zebrafish  we interrogated the function of DNA methylome and its inheritance in early zebrafish development.,,pubmed:34936444,,Dome ctrl scRNA seq rep2,GSM5351823,,source name:embryo|strain:AB|genotype:WT|developmental stage:Dome|tissue:embryo|treatment:microinjection with Rhodamine B and control MO at oogenesis III stage GV,Dome ctrl scRNA seq rep2,All STEM seq datasets were mapped to the danRer7 reference genome by Bismark Krueger and Andrews  2011. Reads were trimmed with cutadaptor Martin  2011 using parameters:   minimum length 20   pair filter=any. Alignments were performed with the following parameters:  N 1  X 600   score min L 0  0.6. Multi mapped reads and PCR duplicates were removed. bismark methylation extractor was used to calculate the DNA methylation level. Total RNA seq data were firstly processed using Trim Galore! with default parameters to trim the adapter containing and low quality reads. The filtered data were then mapped to the zebrafish reference genome danRer7 by STAR version: STAR 2.5.3a modified Dobin et al.  2013 with the following parameters:   outSAMstrandField intronMotif   outSAMattributes All   outSAMunmapped Within   outSAMattrIHstart 0   outWigStrand Stranded   outFilterMultimapNmax 20   twopassMode Basic. The gene expression level was normalized to fragments per kilobase of transcript per million mapped FPKM values using Cufflinks version 2.2.1 Trapnell et al.  2012. The scRNA seq data were processed with Cell Ranger 3.1 for genome alignment danRer10  transcript counting  and gene cell barcode matrix generation. Before the downstream analysis  cells with unique detected genes lower than <2 000 and higher than 6 000 genes were discarded  and cells with high percentage of mitochondrial genes >5% were removed too. Subtypes of cells were identified with Seurat 3.0 through the integrating of control and mKD embryos at dome and shield stages with setting control embryos as reference. To make the data comparable samples  we performed SCTransform normalization separately for each dataset. To further reduce the bais caused by sequencing depth of scRNA seq and percentage of mitochondrial genes  UMI variance and percent.mt were regressed out from SCTransform normalized data matrix by linear model with negative binomial distribution. All ChIP seq reads were aligned to the zebrafish reference genome danRer7 using Bowtie2 version 2.2.2 Langmead and Salzberg  2012 with the parameters –t –q –N 1 –L 25. All unmapped reads  non uniquely mapped reads and PCR duplicates were removed. For downstream analysis  the read counts were normalized by computing the numbers of reads per kilobase of bin per million of reads sequenced RPKM. To visualize the STAR ChIP seq signal in the UCSC genome browser  each read was extended by 250 bp and the coverage for each base was counted. Replicates of STAR ChIP seq were pooled for the downstream analysis. The ATAC seq data were mapped to the zebrafish reference genome danRer7 using Bowtie2 version 2.2.2 Langmead and Salzberg  2012 with the parameters –t –q –N 1 –L 25. All unmapped reads  non uniquely mapped reads and PCR duplicates were removed. For downstream analysis  the read counts were normalized by computing the numbers of reads per kilobase of bin per million of reads sequenced RPKM. To minimize the batch and cell type variation  RPKM values across whole genome were further Z score normalized. To visualize the CUT&RUN signals in the UCSC genome browser  each read was extended by 250 bp and the coverage for each base was counted. Genome build: danRer7 Supplementary files format and content: Matrix for scRNA seq data  gene expression table for total RNA seq data  Bigwig and narrowPeak for ChIP seq and ATAC seq data,embryo,Briefly  on the first day of OMIS Wu et al.  2018b  adult females at 5 mpf 12 mpf were anesthetized in 550 µg/ml tricaine Sigma  Cat A5040 in a petri dish. Then the fish was placed on a damp sponge with specific buffer 5.4 mM KCl  136.8 mM NaCl  4.2 mM NaHCO3  0.44 mM KH2PO4  0.25 mM Na2HPO4  and 0.5% wt/vol BSA. A cut was made on one side of belly to expose the ovary. The diluted MOs were microinjected into each oocyte. Rhodamine B Sigma  Cat R8881 was co injected with MOs as a dye. post injection  the wound on the belly was sewed with a surgical sewing needle carefully and quickly. Once the operation was done  the female was transferred into fish water supplemented with 32 µg/ml tricaine  10 unit/ml penicillin and 10 µg/ml streptomycin HyClone  Cat SV30010. Then the fish was transferred to fish water containing gradually reduced concentrations of tricaine. In the evening of the second day  the female was paired with a wild type male. In the morning of the third day  the pair started to chase and lay fertilized eggs naturally. The injected oocyte derived embryos were identified by co injected dye rhodamine B at very early developmental stages. The injection doses of standard control MO cMO were 5 ng per oocyte in the same assay. MOs were dissolved in RNase free water  and heated to 65°C for 10 min before microinjection.,Cell dissociation protocol was based on a previously described method Farrell et al.  2018 with modifications to adapt it for 10x Genomics platform. Briefly  dnmt1 mKD and control embryos were collected 20 min post fertilization and cultured as mentioned before. Then 15 embryos at dome or shield stages for each sample were transferred into plastic Petri dishes that had previously been coated with 2% agarose and soaked with DMEM/F12 media Gibco/Life Technologies  Cat 11330032 at least 2hrs. Next  embryos at desired stages were dechorionated and deyolked manually by forceps and transferred to a 1.5 ml Eppendorf tube with 50 µl DMEM/F12 media. Dissections were performed for up 15 min. The volume of DMEM/F12 meida containing embryos was adjusted to 200 µl  then cells were mechanically dissociated by flicking the tube 30 times and pipetting mixture 10 times through a 200 µl tip. The volume was adjusted to 1 ml with PBS containing 1.0% BSA  and spun to pellet cells at 300 g for 30s. The supernatant was removed and cells were resuspended in 80 µl of PBS containing 0.1% BSA and 20% Optiprep StemCell  Cat 07820  aiming for a concentration above 100 cells/µl. Cells were then passed through a cell sieve 100 µm for dome stage and 70 µm for shield stage. Single cell RNA seq library was performed with Chromium Next GEM Single Cell kit 10x Genomics  Cat PN 1000121 based on standard protocol. All libraries were sequenced by Illumina Hi Seq 1500 or 2500 or XTen platform according to manufacturer’s instruction.,The wild type AB strain was used in the experiments. The females post OMIS were fed with live adult brine shrimp thrice a day. All embryos were raised in Holtfreter’s solution at 28.5C and staged as described previously Kimmel et al.  1995.,strain:AB|genotype:WT|developmental stage:Dome|tissue:embryo|treatment:microinjection with Rhodamine B and control MO at oogenesis III stage GV,GSM5351823,GSM5351823: Dome ctrl scRNA seq rep2; Danio rerio; RNA Seq,GSM5351823,,1,Cell dissociation protocol was based on a previously described method Farrell et al.  2018 with modifications to adapt it for 10x Genomics platform. Briefly  dnmt1 mKD and control embryos were collected 20 min post fertilization and cultured as mentioned before. Then 15 embryos at dome or shield stages for each sample were transferred into plastic Petri dishes that had previously been coated with 2% agarose and soaked with DMEM/F12 media Gibco/Life Technologies  Cat 11330032 at least 2hrs. Next  embryos at desired stages were dechorionated and deyolked manually by forceps and transferred to a 1.5 ml Eppendorf tube with 50 µl DMEM/F12 media. Dissections were performed for up 15 min. The volume of DMEM/F12 meida containing embryos was adjusted to 200 µl  then cells were mechanically dissociated by flicking the tube 30 times and pipetting mixture 10 times through a 200 µl tip. The volume was adjusted to 1 ml with PBS containing 1.0% BSA  and spun to pellet cells at 300 g for 30s. The supernatant was removed and cells were resuspended in 80 µl of PBS containing 0.1% BSA and 20% Optiprep StemCell  Cat 07820  aiming for a concentration above 100 cells/µl. Cells were then passed through a cell sieve 100 µm for dome stage and 70 µm for shield stage. Single cell RNA seq library was performed with Chromium Next GEM Single Cell kit 10x Genomics  Cat PN 1000121 based on standard protocol. All libraries were sequenced by Illumina Hi Seq 1500 or 2500 or XTen platform according to manufacturer's instruction.,GEO Accession:GSM5351823,RNA-Seq,TRANSCRIPTOMIC,cDNA,PAIRED,ILLUMINA,Illumina HiSeq 1500,,SRP322195,,loader:fastq load.py,Dome_ctrl_scRNA_seq_rep2_2_r1.fq.gz Dome_ctrl_scRNA_seq_rep2_2_r2.fq.gz,fastq fastq,650893500.0,2169645.0,GSM5351823 r2,0:150 1:150,A:162766963;C:121570894;G:127528031;T:239005271;N:22341,150,150,,,162766963,121570894,127528031,239005271,22341,SRX11041468,SRS9110395,SRA1239365,GEO,"THU-PKU Center for Life Sciences, Tsinghua University",2,0.39969,0.87703,0.09623,0.14404,0.94582,0.79494,0.57268,0.54758,150,150,B,B,mate1-mate2 similar by mapping diff,illumina,hiseq_era,unknown,cdna_unspecified,unknown,sc,single_cell_droplet,10x,,China,2021-06-01,Blastula,Embryo,Embryo Imprecise,All anatomical structures
64441,SRR14703452,SRX11041468,SRS9110395,SRP322195,PRJNA734348,Methylome inheritance and enhancer dememorization construct an epigenetic gate safeguarding embryonic programs,GSE175951,Other,Unlike that of mammals  the total DNA methylome of many cold blooded vertebrates is globally inherited from gametes to early embryos. In zebrafish  this is however accompanied by sweeping “dememorization” of enhancers prior to fertilization for sperm and just post fertilization for oocyte  as they undergo full methylation and are not demethylated again until phylotypic stage. The significance of both global methylome inheritance and enhancer dememorization in early embryos remains largely unknown. Adding to the puzzles  the zygotic mutant zebrafish of dnmt1  the major DNA methylation maintenance methyltransferase  surprisingly can develop to term. To solve the role of DNA methylation in early development  we generated zebrafish embryos derived from dnmt1 knocking down oocytes using a recently developed method OMIS microinjection in situ  which successfully eliminated DNA methylation before zygotic genome activation. dnmt1 deficient embryos failed to initiate epiboly and died around gastrulation. This is in part caused by activation of immune response and p53 regulated apoptosis  likely triggered by the derepression of transposable elements. Single cell RNA seq further revealed defective differentiation in these mutants. DNA methylation is also required for the establishment of repressive histone marks H3K27me3 and H2AK119ub. Strikingly  the loss of DNA methylation leads to extensive derepression of somatic genes and enhancers  which acquire ectopic H3K27ac  accessible chromatin  and H3K4me3. These somatic enhancers are preferentially CG rich and are bound by CG containing TFs. By contrast  embryonic enhancers are generally CG poor  methylation insensitive  and are bound by CG less TFs. Hence  the global DNA methylome inheritance is essential for vertebrate early development  and enhancer dememorization resets an epigenetic gate that separates embryonic and somatic programs. Overall design: By employing MethylC seq  total RNA seq  scRNA seq  CUT&RUN  ChIP seq  and ATAC seq in control and dnmt1 mKD embryos at oocyte  256 cell  dome and shield stages in zebrafish  we interrogated the function of DNA methylome and its inheritance in early zebrafish development.,,pubmed:34936444,,Dome ctrl scRNA seq rep2,GSM5351823,,source name:embryo|strain:AB|genotype:WT|developmental stage:Dome|tissue:embryo|treatment:microinjection with Rhodamine B and control MO at oogenesis III stage GV,Dome ctrl scRNA seq rep2,All STEM seq datasets were mapped to the danRer7 reference genome by Bismark Krueger and Andrews  2011. Reads were trimmed with cutadaptor Martin  2011 using parameters:   minimum length 20   pair filter=any. Alignments were performed with the following parameters:  N 1  X 600   score min L 0  0.6. Multi mapped reads and PCR duplicates were removed. bismark methylation extractor was used to calculate the DNA methylation level. Total RNA seq data were firstly processed using Trim Galore! with default parameters to trim the adapter containing and low quality reads. The filtered data were then mapped to the zebrafish reference genome danRer7 by STAR version: STAR 2.5.3a modified Dobin et al.  2013 with the following parameters:   outSAMstrandField intronMotif   outSAMattributes All   outSAMunmapped Within   outSAMattrIHstart 0   outWigStrand Stranded   outFilterMultimapNmax 20   twopassMode Basic. The gene expression level was normalized to fragments per kilobase of transcript per million mapped FPKM values using Cufflinks version 2.2.1 Trapnell et al.  2012. The scRNA seq data were processed with Cell Ranger 3.1 for genome alignment danRer10  transcript counting  and gene cell barcode matrix generation. Before the downstream analysis  cells with unique detected genes lower than <2 000 and higher than 6 000 genes were discarded  and cells with high percentage of mitochondrial genes >5% were removed too. Subtypes of cells were identified with Seurat 3.0 through the integrating of control and mKD embryos at dome and shield stages with setting control embryos as reference. To make the data comparable samples  we performed SCTransform normalization separately for each dataset. To further reduce the bais caused by sequencing depth of scRNA seq and percentage of mitochondrial genes  UMI variance and percent.mt were regressed out from SCTransform normalized data matrix by linear model with negative binomial distribution. All ChIP seq reads were aligned to the zebrafish reference genome danRer7 using Bowtie2 version 2.2.2 Langmead and Salzberg  2012 with the parameters –t –q –N 1 –L 25. All unmapped reads  non uniquely mapped reads and PCR duplicates were removed. For downstream analysis  the read counts were normalized by computing the numbers of reads per kilobase of bin per million of reads sequenced RPKM. To visualize the STAR ChIP seq signal in the UCSC genome browser  each read was extended by 250 bp and the coverage for each base was counted. Replicates of STAR ChIP seq were pooled for the downstream analysis. The ATAC seq data were mapped to the zebrafish reference genome danRer7 using Bowtie2 version 2.2.2 Langmead and Salzberg  2012 with the parameters –t –q –N 1 –L 25. All unmapped reads  non uniquely mapped reads and PCR duplicates were removed. For downstream analysis  the read counts were normalized by computing the numbers of reads per kilobase of bin per million of reads sequenced RPKM. To minimize the batch and cell type variation  RPKM values across whole genome were further Z score normalized. To visualize the CUT&RUN signals in the UCSC genome browser  each read was extended by 250 bp and the coverage for each base was counted. Genome build: danRer7 Supplementary files format and content: Matrix for scRNA seq data  gene expression table for total RNA seq data  Bigwig and narrowPeak for ChIP seq and ATAC seq data,embryo,Briefly  on the first day of OMIS Wu et al.  2018b  adult females at 5 mpf 12 mpf were anesthetized in 550 µg/ml tricaine Sigma  Cat A5040 in a petri dish. Then the fish was placed on a damp sponge with specific buffer 5.4 mM KCl  136.8 mM NaCl  4.2 mM NaHCO3  0.44 mM KH2PO4  0.25 mM Na2HPO4  and 0.5% wt/vol BSA. A cut was made on one side of belly to expose the ovary. The diluted MOs were microinjected into each oocyte. Rhodamine B Sigma  Cat R8881 was co injected with MOs as a dye. post injection  the wound on the belly was sewed with a surgical sewing needle carefully and quickly. Once the operation was done  the female was transferred into fish water supplemented with 32 µg/ml tricaine  10 unit/ml penicillin and 10 µg/ml streptomycin HyClone  Cat SV30010. Then the fish was transferred to fish water containing gradually reduced concentrations of tricaine. In the evening of the second day  the female was paired with a wild type male. In the morning of the third day  the pair started to chase and lay fertilized eggs naturally. The injected oocyte derived embryos were identified by co injected dye rhodamine B at very early developmental stages. The injection doses of standard control MO cMO were 5 ng per oocyte in the same assay. MOs were dissolved in RNase free water  and heated to 65°C for 10 min before microinjection.,Cell dissociation protocol was based on a previously described method Farrell et al.  2018 with modifications to adapt it for 10x Genomics platform. Briefly  dnmt1 mKD and control embryos were collected 20 min post fertilization and cultured as mentioned before. Then 15 embryos at dome or shield stages for each sample were transferred into plastic Petri dishes that had previously been coated with 2% agarose and soaked with DMEM/F12 media Gibco/Life Technologies  Cat 11330032 at least 2hrs. Next  embryos at desired stages were dechorionated and deyolked manually by forceps and transferred to a 1.5 ml Eppendorf tube with 50 µl DMEM/F12 media. Dissections were performed for up 15 min. The volume of DMEM/F12 meida containing embryos was adjusted to 200 µl  then cells were mechanically dissociated by flicking the tube 30 times and pipetting mixture 10 times through a 200 µl tip. The volume was adjusted to 1 ml with PBS containing 1.0% BSA  and spun to pellet cells at 300 g for 30s. The supernatant was removed and cells were resuspended in 80 µl of PBS containing 0.1% BSA and 20% Optiprep StemCell  Cat 07820  aiming for a concentration above 100 cells/µl. Cells were then passed through a cell sieve 100 µm for dome stage and 70 µm for shield stage. Single cell RNA seq library was performed with Chromium Next GEM Single Cell kit 10x Genomics  Cat PN 1000121 based on standard protocol. All libraries were sequenced by Illumina Hi Seq 1500 or 2500 or XTen platform according to manufacturer’s instruction.,The wild type AB strain was used in the experiments. The females post OMIS were fed with live adult brine shrimp thrice a day. All embryos were raised in Holtfreter’s solution at 28.5C and staged as described previously Kimmel et al.  1995.,strain:AB|genotype:WT|developmental stage:Dome|tissue:embryo|treatment:microinjection with Rhodamine B and control MO at oogenesis III stage GV,GSM5351823,GSM5351823: Dome ctrl scRNA seq rep2; Danio rerio; RNA Seq,GSM5351823,,1,Cell dissociation protocol was based on a previously described method Farrell et al.  2018 with modifications to adapt it for 10x Genomics platform. Briefly  dnmt1 mKD and control embryos were collected 20 min post fertilization and cultured as mentioned before. Then 15 embryos at dome or shield stages for each sample were transferred into plastic Petri dishes that had previously been coated with 2% agarose and soaked with DMEM/F12 media Gibco/Life Technologies  Cat 11330032 at least 2hrs. Next  embryos at desired stages were dechorionated and deyolked manually by forceps and transferred to a 1.5 ml Eppendorf tube with 50 µl DMEM/F12 media. Dissections were performed for up 15 min. The volume of DMEM/F12 meida containing embryos was adjusted to 200 µl  then cells were mechanically dissociated by flicking the tube 30 times and pipetting mixture 10 times through a 200 µl tip. The volume was adjusted to 1 ml with PBS containing 1.0% BSA  and spun to pellet cells at 300 g for 30s. The supernatant was removed and cells were resuspended in 80 µl of PBS containing 0.1% BSA and 20% Optiprep StemCell  Cat 07820  aiming for a concentration above 100 cells/µl. Cells were then passed through a cell sieve 100 µm for dome stage and 70 µm for shield stage. Single cell RNA seq library was performed with Chromium Next GEM Single Cell kit 10x Genomics  Cat PN 1000121 based on standard protocol. All libraries were sequenced by Illumina Hi Seq 1500 or 2500 or XTen platform according to manufacturer's instruction.,GEO Accession:GSM5351823,RNA-Seq,TRANSCRIPTOMIC,cDNA,PAIRED,ILLUMINA,Illumina HiSeq 1500,,SRP322195,,loader:fastq load.py,Dome_ctrl_scRNA_seq_rep2_3_r1.fq.gz Dome_ctrl_scRNA_seq_rep2_3_r2.fq.gz,fastq fastq,4967166000.0,16557220.0,GSM5351823 r3,0:150 1:150,A:1244873036;C:927700233;G:972670355;T:1821751756;N:170620,150,150,,,1244873036,927700233,972670355,1821751756,170620,SRX11041468,SRS9110395,SRA1239365,GEO,"THU-PKU Center for Life Sciences, Tsinghua University",2,0.38882,0.87638,0.09482,0.14504,0.94702,0.79498,0.56821,0.54447,150,150,B,B,mate1-mate2 similar by mapping diff,illumina,hiseq_era,unknown,cdna_unspecified,unknown,sc,single_cell_droplet,10x,,China,2021-06-01,Blastula,Embryo,Embryo Imprecise,All anatomical structures
64442,SRR14703453,SRX11041468,SRS9110395,SRP322195,PRJNA734348,Methylome inheritance and enhancer dememorization construct an epigenetic gate safeguarding embryonic programs,GSE175951,Other,Unlike that of mammals  the total DNA methylome of many cold blooded vertebrates is globally inherited from gametes to early embryos. In zebrafish  this is however accompanied by sweeping “dememorization” of enhancers prior to fertilization for sperm and just post fertilization for oocyte  as they undergo full methylation and are not demethylated again until phylotypic stage. The significance of both global methylome inheritance and enhancer dememorization in early embryos remains largely unknown. Adding to the puzzles  the zygotic mutant zebrafish of dnmt1  the major DNA methylation maintenance methyltransferase  surprisingly can develop to term. To solve the role of DNA methylation in early development  we generated zebrafish embryos derived from dnmt1 knocking down oocytes using a recently developed method OMIS microinjection in situ  which successfully eliminated DNA methylation before zygotic genome activation. dnmt1 deficient embryos failed to initiate epiboly and died around gastrulation. This is in part caused by activation of immune response and p53 regulated apoptosis  likely triggered by the derepression of transposable elements. Single cell RNA seq further revealed defective differentiation in these mutants. DNA methylation is also required for the establishment of repressive histone marks H3K27me3 and H2AK119ub. Strikingly  the loss of DNA methylation leads to extensive derepression of somatic genes and enhancers  which acquire ectopic H3K27ac  accessible chromatin  and H3K4me3. These somatic enhancers are preferentially CG rich and are bound by CG containing TFs. By contrast  embryonic enhancers are generally CG poor  methylation insensitive  and are bound by CG less TFs. Hence  the global DNA methylome inheritance is essential for vertebrate early development  and enhancer dememorization resets an epigenetic gate that separates embryonic and somatic programs. Overall design: By employing MethylC seq  total RNA seq  scRNA seq  CUT&RUN  ChIP seq  and ATAC seq in control and dnmt1 mKD embryos at oocyte  256 cell  dome and shield stages in zebrafish  we interrogated the function of DNA methylome and its inheritance in early zebrafish development.,,pubmed:34936444,,Dome ctrl scRNA seq rep2,GSM5351823,,source name:embryo|strain:AB|genotype:WT|developmental stage:Dome|tissue:embryo|treatment:microinjection with Rhodamine B and control MO at oogenesis III stage GV,Dome ctrl scRNA seq rep2,All STEM seq datasets were mapped to the danRer7 reference genome by Bismark Krueger and Andrews  2011. Reads were trimmed with cutadaptor Martin  2011 using parameters:   minimum length 20   pair filter=any. Alignments were performed with the following parameters:  N 1  X 600   score min L 0  0.6. Multi mapped reads and PCR duplicates were removed. bismark methylation extractor was used to calculate the DNA methylation level. Total RNA seq data were firstly processed using Trim Galore! with default parameters to trim the adapter containing and low quality reads. The filtered data were then mapped to the zebrafish reference genome danRer7 by STAR version: STAR 2.5.3a modified Dobin et al.  2013 with the following parameters:   outSAMstrandField intronMotif   outSAMattributes All   outSAMunmapped Within   outSAMattrIHstart 0   outWigStrand Stranded   outFilterMultimapNmax 20   twopassMode Basic. The gene expression level was normalized to fragments per kilobase of transcript per million mapped FPKM values using Cufflinks version 2.2.1 Trapnell et al.  2012. The scRNA seq data were processed with Cell Ranger 3.1 for genome alignment danRer10  transcript counting  and gene cell barcode matrix generation. Before the downstream analysis  cells with unique detected genes lower than <2 000 and higher than 6 000 genes were discarded  and cells with high percentage of mitochondrial genes >5% were removed too. Subtypes of cells were identified with Seurat 3.0 through the integrating of control and mKD embryos at dome and shield stages with setting control embryos as reference. To make the data comparable samples  we performed SCTransform normalization separately for each dataset. To further reduce the bais caused by sequencing depth of scRNA seq and percentage of mitochondrial genes  UMI variance and percent.mt were regressed out from SCTransform normalized data matrix by linear model with negative binomial distribution. All ChIP seq reads were aligned to the zebrafish reference genome danRer7 using Bowtie2 version 2.2.2 Langmead and Salzberg  2012 with the parameters –t –q –N 1 –L 25. All unmapped reads  non uniquely mapped reads and PCR duplicates were removed. For downstream analysis  the read counts were normalized by computing the numbers of reads per kilobase of bin per million of reads sequenced RPKM. To visualize the STAR ChIP seq signal in the UCSC genome browser  each read was extended by 250 bp and the coverage for each base was counted. Replicates of STAR ChIP seq were pooled for the downstream analysis. The ATAC seq data were mapped to the zebrafish reference genome danRer7 using Bowtie2 version 2.2.2 Langmead and Salzberg  2012 with the parameters –t –q –N 1 –L 25. All unmapped reads  non uniquely mapped reads and PCR duplicates were removed. For downstream analysis  the read counts were normalized by computing the numbers of reads per kilobase of bin per million of reads sequenced RPKM. To minimize the batch and cell type variation  RPKM values across whole genome were further Z score normalized. To visualize the CUT&RUN signals in the UCSC genome browser  each read was extended by 250 bp and the coverage for each base was counted. Genome build: danRer7 Supplementary files format and content: Matrix for scRNA seq data  gene expression table for total RNA seq data  Bigwig and narrowPeak for ChIP seq and ATAC seq data,embryo,Briefly  on the first day of OMIS Wu et al.  2018b  adult females at 5 mpf 12 mpf were anesthetized in 550 µg/ml tricaine Sigma  Cat A5040 in a petri dish. Then the fish was placed on a damp sponge with specific buffer 5.4 mM KCl  136.8 mM NaCl  4.2 mM NaHCO3  0.44 mM KH2PO4  0.25 mM Na2HPO4  and 0.5% wt/vol BSA. A cut was made on one side of belly to expose the ovary. The diluted MOs were microinjected into each oocyte. Rhodamine B Sigma  Cat R8881 was co injected with MOs as a dye. post injection  the wound on the belly was sewed with a surgical sewing needle carefully and quickly. Once the operation was done  the female was transferred into fish water supplemented with 32 µg/ml tricaine  10 unit/ml penicillin and 10 µg/ml streptomycin HyClone  Cat SV30010. Then the fish was transferred to fish water containing gradually reduced concentrations of tricaine. In the evening of the second day  the female was paired with a wild type male. In the morning of the third day  the pair started to chase and lay fertilized eggs naturally. The injected oocyte derived embryos were identified by co injected dye rhodamine B at very early developmental stages. The injection doses of standard control MO cMO were 5 ng per oocyte in the same assay. MOs were dissolved in RNase free water  and heated to 65°C for 10 min before microinjection.,Cell dissociation protocol was based on a previously described method Farrell et al.  2018 with modifications to adapt it for 10x Genomics platform. Briefly  dnmt1 mKD and control embryos were collected 20 min post fertilization and cultured as mentioned before. Then 15 embryos at dome or shield stages for each sample were transferred into plastic Petri dishes that had previously been coated with 2% agarose and soaked with DMEM/F12 media Gibco/Life Technologies  Cat 11330032 at least 2hrs. Next  embryos at desired stages were dechorionated and deyolked manually by forceps and transferred to a 1.5 ml Eppendorf tube with 50 µl DMEM/F12 media. Dissections were performed for up 15 min. The volume of DMEM/F12 meida containing embryos was adjusted to 200 µl  then cells were mechanically dissociated by flicking the tube 30 times and pipetting mixture 10 times through a 200 µl tip. The volume was adjusted to 1 ml with PBS containing 1.0% BSA  and spun to pellet cells at 300 g for 30s. The supernatant was removed and cells were resuspended in 80 µl of PBS containing 0.1% BSA and 20% Optiprep StemCell  Cat 07820  aiming for a concentration above 100 cells/µl. Cells were then passed through a cell sieve 100 µm for dome stage and 70 µm for shield stage. Single cell RNA seq library was performed with Chromium Next GEM Single Cell kit 10x Genomics  Cat PN 1000121 based on standard protocol. All libraries were sequenced by Illumina Hi Seq 1500 or 2500 or XTen platform according to manufacturer’s instruction.,The wild type AB strain was used in the experiments. The females post OMIS were fed with live adult brine shrimp thrice a day. All embryos were raised in Holtfreter’s solution at 28.5C and staged as described previously Kimmel et al.  1995.,strain:AB|genotype:WT|developmental stage:Dome|tissue:embryo|treatment:microinjection with Rhodamine B and control MO at oogenesis III stage GV,GSM5351823,GSM5351823: Dome ctrl scRNA seq rep2; Danio rerio; RNA Seq,GSM5351823,,1,Cell dissociation protocol was based on a previously described method Farrell et al.  2018 with modifications to adapt it for 10x Genomics platform. Briefly  dnmt1 mKD and control embryos were collected 20 min post fertilization and cultured as mentioned before. Then 15 embryos at dome or shield stages for each sample were transferred into plastic Petri dishes that had previously been coated with 2% agarose and soaked with DMEM/F12 media Gibco/Life Technologies  Cat 11330032 at least 2hrs. Next  embryos at desired stages were dechorionated and deyolked manually by forceps and transferred to a 1.5 ml Eppendorf tube with 50 µl DMEM/F12 media. Dissections were performed for up 15 min. The volume of DMEM/F12 meida containing embryos was adjusted to 200 µl  then cells were mechanically dissociated by flicking the tube 30 times and pipetting mixture 10 times through a 200 µl tip. The volume was adjusted to 1 ml with PBS containing 1.0% BSA  and spun to pellet cells at 300 g for 30s. The supernatant was removed and cells were resuspended in 80 µl of PBS containing 0.1% BSA and 20% Optiprep StemCell  Cat 07820  aiming for a concentration above 100 cells/µl. Cells were then passed through a cell sieve 100 µm for dome stage and 70 µm for shield stage. Single cell RNA seq library was performed with Chromium Next GEM Single Cell kit 10x Genomics  Cat PN 1000121 based on standard protocol. All libraries were sequenced by Illumina Hi Seq 1500 or 2500 or XTen platform according to manufacturer's instruction.,GEO Accession:GSM5351823,RNA-Seq,TRANSCRIPTOMIC,cDNA,PAIRED,ILLUMINA,Illumina HiSeq 1500,,SRP322195,,loader:fastq load.py,Dome_ctrl_scRNA_seq_rep2_4_r1.fq.gz Dome_ctrl_scRNA_seq_rep2_4_r2.fq.gz,fastq fastq,4674495600.0,15581652.0,GSM5351823 r4,0:150 1:150,A:1169120315;C:876915369;G:918802081;T:1709495035;N:162800,150,150,,,1169120315,876915369,918802081,1709495035,162800,SRX11041468,SRS9110395,SRA1239365,GEO,"THU-PKU Center for Life Sciences, Tsinghua University",2,0.40306,0.87902,0.09932,0.14392,0.94627,0.79632,0.56259,0.54307,150,150,B,B,mate1-mate2 similar by mapping diff,illumina,hiseq_era,unknown,cdna_unspecified,unknown,sc,single_cell_droplet,10x,,China,2021-06-01,Blastula,Embryo,Embryo Imprecise,All anatomical structures
64443,SRR14703446,SRX11041467,SRS9110397,SRP322195,PRJNA734348,Methylome inheritance and enhancer dememorization construct an epigenetic gate safeguarding embryonic programs,GSE175951,Other,Unlike that of mammals  the total DNA methylome of many cold blooded vertebrates is globally inherited from gametes to early embryos. In zebrafish  this is however accompanied by sweeping “dememorization” of enhancers prior to fertilization for sperm and just post fertilization for oocyte  as they undergo full methylation and are not demethylated again until phylotypic stage. The significance of both global methylome inheritance and enhancer dememorization in early embryos remains largely unknown. Adding to the puzzles  the zygotic mutant zebrafish of dnmt1  the major DNA methylation maintenance methyltransferase  surprisingly can develop to term. To solve the role of DNA methylation in early development  we generated zebrafish embryos derived from dnmt1 knocking down oocytes using a recently developed method OMIS microinjection in situ  which successfully eliminated DNA methylation before zygotic genome activation. dnmt1 deficient embryos failed to initiate epiboly and died around gastrulation. This is in part caused by activation of immune response and p53 regulated apoptosis  likely triggered by the derepression of transposable elements. Single cell RNA seq further revealed defective differentiation in these mutants. DNA methylation is also required for the establishment of repressive histone marks H3K27me3 and H2AK119ub. Strikingly  the loss of DNA methylation leads to extensive derepression of somatic genes and enhancers  which acquire ectopic H3K27ac  accessible chromatin  and H3K4me3. These somatic enhancers are preferentially CG rich and are bound by CG containing TFs. By contrast  embryonic enhancers are generally CG poor  methylation insensitive  and are bound by CG less TFs. Hence  the global DNA methylome inheritance is essential for vertebrate early development  and enhancer dememorization resets an epigenetic gate that separates embryonic and somatic programs. Overall design: By employing MethylC seq  total RNA seq  scRNA seq  CUT&RUN  ChIP seq  and ATAC seq in control and dnmt1 mKD embryos at oocyte  256 cell  dome and shield stages in zebrafish  we interrogated the function of DNA methylome and its inheritance in early zebrafish development.,,pubmed:34936444,,Dome ctrl scRNA seq rep1,GSM5351822,,source name:embryo|strain:AB|genotype:WT|developmental stage:Dome|tissue:embryo|treatment:microinjection with Rhodamine B and control MO at oogenesis III stage GV,Dome ctrl scRNA seq rep1,All STEM seq datasets were mapped to the danRer7 reference genome by Bismark Krueger and Andrews  2011. Reads were trimmed with cutadaptor Martin  2011 using parameters:   minimum length 20   pair filter=any. Alignments were performed with the following parameters:  N 1  X 600   score min L 0  0.6. Multi mapped reads and PCR duplicates were removed. bismark methylation extractor was used to calculate the DNA methylation level. Total RNA seq data were firstly processed using Trim Galore! with default parameters to trim the adapter containing and low quality reads. The filtered data were then mapped to the zebrafish reference genome danRer7 by STAR version: STAR 2.5.3a modified Dobin et al.  2013 with the following parameters:   outSAMstrandField intronMotif   outSAMattributes All   outSAMunmapped Within   outSAMattrIHstart 0   outWigStrand Stranded   outFilterMultimapNmax 20   twopassMode Basic. The gene expression level was normalized to fragments per kilobase of transcript per million mapped FPKM values using Cufflinks version 2.2.1 Trapnell et al.  2012. The scRNA seq data were processed with Cell Ranger 3.1 for genome alignment danRer10  transcript counting  and gene cell barcode matrix generation. Before the downstream analysis  cells with unique detected genes lower than <2 000 and higher than 6 000 genes were discarded  and cells with high percentage of mitochondrial genes >5% were removed too. Subtypes of cells were identified with Seurat 3.0 through the integrating of control and mKD embryos at dome and shield stages with setting control embryos as reference. To make the data comparable samples  we performed SCTransform normalization separately for each dataset. To further reduce the bais caused by sequencing depth of scRNA seq and percentage of mitochondrial genes  UMI variance and percent.mt were regressed out from SCTransform normalized data matrix by linear model with negative binomial distribution. All ChIP seq reads were aligned to the zebrafish reference genome danRer7 using Bowtie2 version 2.2.2 Langmead and Salzberg  2012 with the parameters –t –q –N 1 –L 25. All unmapped reads  non uniquely mapped reads and PCR duplicates were removed. For downstream analysis  the read counts were normalized by computing the numbers of reads per kilobase of bin per million of reads sequenced RPKM. To visualize the STAR ChIP seq signal in the UCSC genome browser  each read was extended by 250 bp and the coverage for each base was counted. Replicates of STAR ChIP seq were pooled for the downstream analysis. The ATAC seq data were mapped to the zebrafish reference genome danRer7 using Bowtie2 version 2.2.2 Langmead and Salzberg  2012 with the parameters –t –q –N 1 –L 25. All unmapped reads  non uniquely mapped reads and PCR duplicates were removed. For downstream analysis  the read counts were normalized by computing the numbers of reads per kilobase of bin per million of reads sequenced RPKM. To minimize the batch and cell type variation  RPKM values across whole genome were further Z score normalized. To visualize the CUT&RUN signals in the UCSC genome browser  each read was extended by 250 bp and the coverage for each base was counted. Genome build: danRer7 Supplementary files format and content: Matrix for scRNA seq data  gene expression table for total RNA seq data  Bigwig and narrowPeak for ChIP seq and ATAC seq data,embryo,Briefly  on the first day of OMIS Wu et al.  2018b  adult females at 5 mpf 12 mpf were anesthetized in 550 µg/ml tricaine Sigma  Cat A5040 in a petri dish. Then the fish was placed on a damp sponge with specific buffer 5.4 mM KCl  136.8 mM NaCl  4.2 mM NaHCO3  0.44 mM KH2PO4  0.25 mM Na2HPO4  and 0.5% wt/vol BSA. A cut was made on one side of belly to expose the ovary. The diluted MOs were microinjected into each oocyte. Rhodamine B Sigma  Cat R8881 was co injected with MOs as a dye. post injection  the wound on the belly was sewed with a surgical sewing needle carefully and quickly. Once the operation was done  the female was transferred into fish water supplemented with 32 µg/ml tricaine  10 unit/ml penicillin and 10 µg/ml streptomycin HyClone  Cat SV30010. Then the fish was transferred to fish water containing gradually reduced concentrations of tricaine. In the evening of the second day  the female was paired with a wild type male. In the morning of the third day  the pair started to chase and lay fertilized eggs naturally. The injected oocyte derived embryos were identified by co injected dye rhodamine B at very early developmental stages. The injection doses of standard control MO cMO were 5 ng per oocyte in the same assay. MOs were dissolved in RNase free water  and heated to 65°C for 10 min before microinjection.,Cell dissociation protocol was based on a previously described method Farrell et al.  2018 with modifications to adapt it for 10x Genomics platform. Briefly  dnmt1 mKD and control embryos were collected 20 min post fertilization and cultured as mentioned before. Then 15 embryos at dome or shield stages for each sample were transferred into plastic Petri dishes that had previously been coated with 2% agarose and soaked with DMEM/F12 media Gibco/Life Technologies  Cat 11330032 at least 2hrs. Next  embryos at desired stages were dechorionated and deyolked manually by forceps and transferred to a 1.5 ml Eppendorf tube with 50 µl DMEM/F12 media. Dissections were performed for up 15 min. The volume of DMEM/F12 meida containing embryos was adjusted to 200 µl  then cells were mechanically dissociated by flicking the tube 30 times and pipetting mixture 10 times through a 200 µl tip. The volume was adjusted to 1 ml with PBS containing 1.0% BSA  and spun to pellet cells at 300 g for 30s. The supernatant was removed and cells were resuspended in 80 µl of PBS containing 0.1% BSA and 20% Optiprep StemCell  Cat 07820  aiming for a concentration above 100 cells/µl. Cells were then passed through a cell sieve 100 µm for dome stage and 70 µm for shield stage. Single cell RNA seq library was performed with Chromium Next GEM Single Cell kit 10x Genomics  Cat PN 1000121 based on standard protocol. All libraries were sequenced by Illumina Hi Seq 1500 or 2500 or XTen platform according to manufacturer’s instruction.,The wild type AB strain was used in the experiments. The females post OMIS were fed with live adult brine shrimp thrice a day. All embryos were raised in Holtfreter’s solution at 28.5C and staged as described previously Kimmel et al.  1995.,strain:AB|genotype:WT|developmental stage:Dome|tissue:embryo|treatment:microinjection with Rhodamine B and control MO at oogenesis III stage GV,GSM5351822,GSM5351822: Dome ctrl scRNA seq rep1; Danio rerio; RNA Seq,GSM5351822,,1,Cell dissociation protocol was based on a previously described method Farrell et al.  2018 with modifications to adapt it for 10x Genomics platform. Briefly  dnmt1 mKD and control embryos were collected 20 min post fertilization and cultured as mentioned before. Then 15 embryos at dome or shield stages for each sample were transferred into plastic Petri dishes that had previously been coated with 2% agarose and soaked with DMEM/F12 media Gibco/Life Technologies  Cat 11330032 at least 2hrs. Next  embryos at desired stages were dechorionated and deyolked manually by forceps and transferred to a 1.5 ml Eppendorf tube with 50 µl DMEM/F12 media. Dissections were performed for up 15 min. The volume of DMEM/F12 meida containing embryos was adjusted to 200 µl  then cells were mechanically dissociated by flicking the tube 30 times and pipetting mixture 10 times through a 200 µl tip. The volume was adjusted to 1 ml with PBS containing 1.0% BSA  and spun to pellet cells at 300 g for 30s. The supernatant was removed and cells were resuspended in 80 µl of PBS containing 0.1% BSA and 20% Optiprep StemCell  Cat 07820  aiming for a concentration above 100 cells/µl. Cells were then passed through a cell sieve 100 µm for dome stage and 70 µm for shield stage. Single cell RNA seq library was performed with Chromium Next GEM Single Cell kit 10x Genomics  Cat PN 1000121 based on standard protocol. All libraries were sequenced by Illumina Hi Seq 1500 or 2500 or XTen platform according to manufacturer's instruction.,GEO Accession:GSM5351822,RNA-Seq,TRANSCRIPTOMIC,cDNA,PAIRED,ILLUMINA,HiSeq X Ten,,SRP322195,,loader:fastq load.py,Dome_ctrl_scRNA_seq_rep1_1_r1.fq.gz Dome_ctrl_scRNA_seq_rep1_1_r2.fq.gz,fastq fastq,9940355100.0,33134517.0,GSM5351822 r1,0:150 1:150,A:2457125197;C:1850376601;G:1936367439;T:3696137056;N:348807,150,150,,,2457125197,1850376601,1936367439,3696137056,348807,SRX11041467,SRS9110397,SRA1239365,GEO,"THU-PKU Center for Life Sciences, Tsinghua University",2,0.34261,0.88683,0.08556,0.08811,0.9516,0.79054,0.59718,0.54828,150,150,T,B,mate1 technical by mapping diff,illumina,hiseq_era,unknown,cdna_unspecified,unknown,sc,single_cell_droplet,10x,,China,2021-06-01,Blastula,Embryo,Embryo Imprecise,All anatomical structures
64444,SRR14703447,SRX11041467,SRS9110397,SRP322195,PRJNA734348,Methylome inheritance and enhancer dememorization construct an epigenetic gate safeguarding embryonic programs,GSE175951,Other,Unlike that of mammals  the total DNA methylome of many cold blooded vertebrates is globally inherited from gametes to early embryos. In zebrafish  this is however accompanied by sweeping “dememorization” of enhancers prior to fertilization for sperm and just post fertilization for oocyte  as they undergo full methylation and are not demethylated again until phylotypic stage. The significance of both global methylome inheritance and enhancer dememorization in early embryos remains largely unknown. Adding to the puzzles  the zygotic mutant zebrafish of dnmt1  the major DNA methylation maintenance methyltransferase  surprisingly can develop to term. To solve the role of DNA methylation in early development  we generated zebrafish embryos derived from dnmt1 knocking down oocytes using a recently developed method OMIS microinjection in situ  which successfully eliminated DNA methylation before zygotic genome activation. dnmt1 deficient embryos failed to initiate epiboly and died around gastrulation. This is in part caused by activation of immune response and p53 regulated apoptosis  likely triggered by the derepression of transposable elements. Single cell RNA seq further revealed defective differentiation in these mutants. DNA methylation is also required for the establishment of repressive histone marks H3K27me3 and H2AK119ub. Strikingly  the loss of DNA methylation leads to extensive derepression of somatic genes and enhancers  which acquire ectopic H3K27ac  accessible chromatin  and H3K4me3. These somatic enhancers are preferentially CG rich and are bound by CG containing TFs. By contrast  embryonic enhancers are generally CG poor  methylation insensitive  and are bound by CG less TFs. Hence  the global DNA methylome inheritance is essential for vertebrate early development  and enhancer dememorization resets an epigenetic gate that separates embryonic and somatic programs. Overall design: By employing MethylC seq  total RNA seq  scRNA seq  CUT&RUN  ChIP seq  and ATAC seq in control and dnmt1 mKD embryos at oocyte  256 cell  dome and shield stages in zebrafish  we interrogated the function of DNA methylome and its inheritance in early zebrafish development.,,pubmed:34936444,,Dome ctrl scRNA seq rep1,GSM5351822,,source name:embryo|strain:AB|genotype:WT|developmental stage:Dome|tissue:embryo|treatment:microinjection with Rhodamine B and control MO at oogenesis III stage GV,Dome ctrl scRNA seq rep1,All STEM seq datasets were mapped to the danRer7 reference genome by Bismark Krueger and Andrews  2011. Reads were trimmed with cutadaptor Martin  2011 using parameters:   minimum length 20   pair filter=any. Alignments were performed with the following parameters:  N 1  X 600   score min L 0  0.6. Multi mapped reads and PCR duplicates were removed. bismark methylation extractor was used to calculate the DNA methylation level. Total RNA seq data were firstly processed using Trim Galore! with default parameters to trim the adapter containing and low quality reads. The filtered data were then mapped to the zebrafish reference genome danRer7 by STAR version: STAR 2.5.3a modified Dobin et al.  2013 with the following parameters:   outSAMstrandField intronMotif   outSAMattributes All   outSAMunmapped Within   outSAMattrIHstart 0   outWigStrand Stranded   outFilterMultimapNmax 20   twopassMode Basic. The gene expression level was normalized to fragments per kilobase of transcript per million mapped FPKM values using Cufflinks version 2.2.1 Trapnell et al.  2012. The scRNA seq data were processed with Cell Ranger 3.1 for genome alignment danRer10  transcript counting  and gene cell barcode matrix generation. Before the downstream analysis  cells with unique detected genes lower than <2 000 and higher than 6 000 genes were discarded  and cells with high percentage of mitochondrial genes >5% were removed too. Subtypes of cells were identified with Seurat 3.0 through the integrating of control and mKD embryos at dome and shield stages with setting control embryos as reference. To make the data comparable samples  we performed SCTransform normalization separately for each dataset. To further reduce the bais caused by sequencing depth of scRNA seq and percentage of mitochondrial genes  UMI variance and percent.mt were regressed out from SCTransform normalized data matrix by linear model with negative binomial distribution. All ChIP seq reads were aligned to the zebrafish reference genome danRer7 using Bowtie2 version 2.2.2 Langmead and Salzberg  2012 with the parameters –t –q –N 1 –L 25. All unmapped reads  non uniquely mapped reads and PCR duplicates were removed. For downstream analysis  the read counts were normalized by computing the numbers of reads per kilobase of bin per million of reads sequenced RPKM. To visualize the STAR ChIP seq signal in the UCSC genome browser  each read was extended by 250 bp and the coverage for each base was counted. Replicates of STAR ChIP seq were pooled for the downstream analysis. The ATAC seq data were mapped to the zebrafish reference genome danRer7 using Bowtie2 version 2.2.2 Langmead and Salzberg  2012 with the parameters –t –q –N 1 –L 25. All unmapped reads  non uniquely mapped reads and PCR duplicates were removed. For downstream analysis  the read counts were normalized by computing the numbers of reads per kilobase of bin per million of reads sequenced RPKM. To minimize the batch and cell type variation  RPKM values across whole genome were further Z score normalized. To visualize the CUT&RUN signals in the UCSC genome browser  each read was extended by 250 bp and the coverage for each base was counted. Genome build: danRer7 Supplementary files format and content: Matrix for scRNA seq data  gene expression table for total RNA seq data  Bigwig and narrowPeak for ChIP seq and ATAC seq data,embryo,Briefly  on the first day of OMIS Wu et al.  2018b  adult females at 5 mpf 12 mpf were anesthetized in 550 µg/ml tricaine Sigma  Cat A5040 in a petri dish. Then the fish was placed on a damp sponge with specific buffer 5.4 mM KCl  136.8 mM NaCl  4.2 mM NaHCO3  0.44 mM KH2PO4  0.25 mM Na2HPO4  and 0.5% wt/vol BSA. A cut was made on one side of belly to expose the ovary. The diluted MOs were microinjected into each oocyte. Rhodamine B Sigma  Cat R8881 was co injected with MOs as a dye. post injection  the wound on the belly was sewed with a surgical sewing needle carefully and quickly. Once the operation was done  the female was transferred into fish water supplemented with 32 µg/ml tricaine  10 unit/ml penicillin and 10 µg/ml streptomycin HyClone  Cat SV30010. Then the fish was transferred to fish water containing gradually reduced concentrations of tricaine. In the evening of the second day  the female was paired with a wild type male. In the morning of the third day  the pair started to chase and lay fertilized eggs naturally. The injected oocyte derived embryos were identified by co injected dye rhodamine B at very early developmental stages. The injection doses of standard control MO cMO were 5 ng per oocyte in the same assay. MOs were dissolved in RNase free water  and heated to 65°C for 10 min before microinjection.,Cell dissociation protocol was based on a previously described method Farrell et al.  2018 with modifications to adapt it for 10x Genomics platform. Briefly  dnmt1 mKD and control embryos were collected 20 min post fertilization and cultured as mentioned before. Then 15 embryos at dome or shield stages for each sample were transferred into plastic Petri dishes that had previously been coated with 2% agarose and soaked with DMEM/F12 media Gibco/Life Technologies  Cat 11330032 at least 2hrs. Next  embryos at desired stages were dechorionated and deyolked manually by forceps and transferred to a 1.5 ml Eppendorf tube with 50 µl DMEM/F12 media. Dissections were performed for up 15 min. The volume of DMEM/F12 meida containing embryos was adjusted to 200 µl  then cells were mechanically dissociated by flicking the tube 30 times and pipetting mixture 10 times through a 200 µl tip. The volume was adjusted to 1 ml with PBS containing 1.0% BSA  and spun to pellet cells at 300 g for 30s. The supernatant was removed and cells were resuspended in 80 µl of PBS containing 0.1% BSA and 20% Optiprep StemCell  Cat 07820  aiming for a concentration above 100 cells/µl. Cells were then passed through a cell sieve 100 µm for dome stage and 70 µm for shield stage. Single cell RNA seq library was performed with Chromium Next GEM Single Cell kit 10x Genomics  Cat PN 1000121 based on standard protocol. All libraries were sequenced by Illumina Hi Seq 1500 or 2500 or XTen platform according to manufacturer’s instruction.,The wild type AB strain was used in the experiments. The females post OMIS were fed with live adult brine shrimp thrice a day. All embryos were raised in Holtfreter’s solution at 28.5C and staged as described previously Kimmel et al.  1995.,strain:AB|genotype:WT|developmental stage:Dome|tissue:embryo|treatment:microinjection with Rhodamine B and control MO at oogenesis III stage GV,GSM5351822,GSM5351822: Dome ctrl scRNA seq rep1; Danio rerio; RNA Seq,GSM5351822,,1,Cell dissociation protocol was based on a previously described method Farrell et al.  2018 with modifications to adapt it for 10x Genomics platform. Briefly  dnmt1 mKD and control embryos were collected 20 min post fertilization and cultured as mentioned before. Then 15 embryos at dome or shield stages for each sample were transferred into plastic Petri dishes that had previously been coated with 2% agarose and soaked with DMEM/F12 media Gibco/Life Technologies  Cat 11330032 at least 2hrs. Next  embryos at desired stages were dechorionated and deyolked manually by forceps and transferred to a 1.5 ml Eppendorf tube with 50 µl DMEM/F12 media. Dissections were performed for up 15 min. The volume of DMEM/F12 meida containing embryos was adjusted to 200 µl  then cells were mechanically dissociated by flicking the tube 30 times and pipetting mixture 10 times through a 200 µl tip. The volume was adjusted to 1 ml with PBS containing 1.0% BSA  and spun to pellet cells at 300 g for 30s. The supernatant was removed and cells were resuspended in 80 µl of PBS containing 0.1% BSA and 20% Optiprep StemCell  Cat 07820  aiming for a concentration above 100 cells/µl. Cells were then passed through a cell sieve 100 µm for dome stage and 70 µm for shield stage. Single cell RNA seq library was performed with Chromium Next GEM Single Cell kit 10x Genomics  Cat PN 1000121 based on standard protocol. All libraries were sequenced by Illumina Hi Seq 1500 or 2500 or XTen platform according to manufacturer's instruction.,GEO Accession:GSM5351822,RNA-Seq,TRANSCRIPTOMIC,cDNA,PAIRED,ILLUMINA,HiSeq X Ten,,SRP322195,,loader:fastq load.py,Dome_ctrl_scRNA_seq_rep1_2_r1.fq.gz Dome_ctrl_scRNA_seq_rep1_2_r2.fq.gz,fastq fastq,9132461400.0,30441538.0,GSM5351822 r2,0:150 1:150,A:2259394050;C:1696157400;G:1777473786;T:3399114425;N:321739,150,150,,,2259394050,1696157400,1777473786,3399114425,321739,SRX11041467,SRS9110397,SRA1239365,GEO,"THU-PKU Center for Life Sciences, Tsinghua University",2,0.33316,0.88826,0.08233,0.08988,0.95266,0.78875,0.59482,0.54173,150,150,T,B,mate1 technical by mapping diff,illumina,hiseq_era,unknown,cdna_unspecified,unknown,sc,single_cell_droplet,10x,,China,2021-06-01,Blastula,Embryo,Embryo Imprecise,All anatomical structures
64445,SRR14703448,SRX11041467,SRS9110397,SRP322195,PRJNA734348,Methylome inheritance and enhancer dememorization construct an epigenetic gate safeguarding embryonic programs,GSE175951,Other,Unlike that of mammals  the total DNA methylome of many cold blooded vertebrates is globally inherited from gametes to early embryos. In zebrafish  this is however accompanied by sweeping “dememorization” of enhancers prior to fertilization for sperm and just post fertilization for oocyte  as they undergo full methylation and are not demethylated again until phylotypic stage. The significance of both global methylome inheritance and enhancer dememorization in early embryos remains largely unknown. Adding to the puzzles  the zygotic mutant zebrafish of dnmt1  the major DNA methylation maintenance methyltransferase  surprisingly can develop to term. To solve the role of DNA methylation in early development  we generated zebrafish embryos derived from dnmt1 knocking down oocytes using a recently developed method OMIS microinjection in situ  which successfully eliminated DNA methylation before zygotic genome activation. dnmt1 deficient embryos failed to initiate epiboly and died around gastrulation. This is in part caused by activation of immune response and p53 regulated apoptosis  likely triggered by the derepression of transposable elements. Single cell RNA seq further revealed defective differentiation in these mutants. DNA methylation is also required for the establishment of repressive histone marks H3K27me3 and H2AK119ub. Strikingly  the loss of DNA methylation leads to extensive derepression of somatic genes and enhancers  which acquire ectopic H3K27ac  accessible chromatin  and H3K4me3. These somatic enhancers are preferentially CG rich and are bound by CG containing TFs. By contrast  embryonic enhancers are generally CG poor  methylation insensitive  and are bound by CG less TFs. Hence  the global DNA methylome inheritance is essential for vertebrate early development  and enhancer dememorization resets an epigenetic gate that separates embryonic and somatic programs. Overall design: By employing MethylC seq  total RNA seq  scRNA seq  CUT&RUN  ChIP seq  and ATAC seq in control and dnmt1 mKD embryos at oocyte  256 cell  dome and shield stages in zebrafish  we interrogated the function of DNA methylome and its inheritance in early zebrafish development.,,pubmed:34936444,,Dome ctrl scRNA seq rep1,GSM5351822,,source name:embryo|strain:AB|genotype:WT|developmental stage:Dome|tissue:embryo|treatment:microinjection with Rhodamine B and control MO at oogenesis III stage GV,Dome ctrl scRNA seq rep1,All STEM seq datasets were mapped to the danRer7 reference genome by Bismark Krueger and Andrews  2011. Reads were trimmed with cutadaptor Martin  2011 using parameters:   minimum length 20   pair filter=any. Alignments were performed with the following parameters:  N 1  X 600   score min L 0  0.6. Multi mapped reads and PCR duplicates were removed. bismark methylation extractor was used to calculate the DNA methylation level. Total RNA seq data were firstly processed using Trim Galore! with default parameters to trim the adapter containing and low quality reads. The filtered data were then mapped to the zebrafish reference genome danRer7 by STAR version: STAR 2.5.3a modified Dobin et al.  2013 with the following parameters:   outSAMstrandField intronMotif   outSAMattributes All   outSAMunmapped Within   outSAMattrIHstart 0   outWigStrand Stranded   outFilterMultimapNmax 20   twopassMode Basic. The gene expression level was normalized to fragments per kilobase of transcript per million mapped FPKM values using Cufflinks version 2.2.1 Trapnell et al.  2012. The scRNA seq data were processed with Cell Ranger 3.1 for genome alignment danRer10  transcript counting  and gene cell barcode matrix generation. Before the downstream analysis  cells with unique detected genes lower than <2 000 and higher than 6 000 genes were discarded  and cells with high percentage of mitochondrial genes >5% were removed too. Subtypes of cells were identified with Seurat 3.0 through the integrating of control and mKD embryos at dome and shield stages with setting control embryos as reference. To make the data comparable samples  we performed SCTransform normalization separately for each dataset. To further reduce the bais caused by sequencing depth of scRNA seq and percentage of mitochondrial genes  UMI variance and percent.mt were regressed out from SCTransform normalized data matrix by linear model with negative binomial distribution. All ChIP seq reads were aligned to the zebrafish reference genome danRer7 using Bowtie2 version 2.2.2 Langmead and Salzberg  2012 with the parameters –t –q –N 1 –L 25. All unmapped reads  non uniquely mapped reads and PCR duplicates were removed. For downstream analysis  the read counts were normalized by computing the numbers of reads per kilobase of bin per million of reads sequenced RPKM. To visualize the STAR ChIP seq signal in the UCSC genome browser  each read was extended by 250 bp and the coverage for each base was counted. Replicates of STAR ChIP seq were pooled for the downstream analysis. The ATAC seq data were mapped to the zebrafish reference genome danRer7 using Bowtie2 version 2.2.2 Langmead and Salzberg  2012 with the parameters –t –q –N 1 –L 25. All unmapped reads  non uniquely mapped reads and PCR duplicates were removed. For downstream analysis  the read counts were normalized by computing the numbers of reads per kilobase of bin per million of reads sequenced RPKM. To minimize the batch and cell type variation  RPKM values across whole genome were further Z score normalized. To visualize the CUT&RUN signals in the UCSC genome browser  each read was extended by 250 bp and the coverage for each base was counted. Genome build: danRer7 Supplementary files format and content: Matrix for scRNA seq data  gene expression table for total RNA seq data  Bigwig and narrowPeak for ChIP seq and ATAC seq data,embryo,Briefly  on the first day of OMIS Wu et al.  2018b  adult females at 5 mpf 12 mpf were anesthetized in 550 µg/ml tricaine Sigma  Cat A5040 in a petri dish. Then the fish was placed on a damp sponge with specific buffer 5.4 mM KCl  136.8 mM NaCl  4.2 mM NaHCO3  0.44 mM KH2PO4  0.25 mM Na2HPO4  and 0.5% wt/vol BSA. A cut was made on one side of belly to expose the ovary. The diluted MOs were microinjected into each oocyte. Rhodamine B Sigma  Cat R8881 was co injected with MOs as a dye. post injection  the wound on the belly was sewed with a surgical sewing needle carefully and quickly. Once the operation was done  the female was transferred into fish water supplemented with 32 µg/ml tricaine  10 unit/ml penicillin and 10 µg/ml streptomycin HyClone  Cat SV30010. Then the fish was transferred to fish water containing gradually reduced concentrations of tricaine. In the evening of the second day  the female was paired with a wild type male. In the morning of the third day  the pair started to chase and lay fertilized eggs naturally. The injected oocyte derived embryos were identified by co injected dye rhodamine B at very early developmental stages. The injection doses of standard control MO cMO were 5 ng per oocyte in the same assay. MOs were dissolved in RNase free water  and heated to 65°C for 10 min before microinjection.,Cell dissociation protocol was based on a previously described method Farrell et al.  2018 with modifications to adapt it for 10x Genomics platform. Briefly  dnmt1 mKD and control embryos were collected 20 min post fertilization and cultured as mentioned before. Then 15 embryos at dome or shield stages for each sample were transferred into plastic Petri dishes that had previously been coated with 2% agarose and soaked with DMEM/F12 media Gibco/Life Technologies  Cat 11330032 at least 2hrs. Next  embryos at desired stages were dechorionated and deyolked manually by forceps and transferred to a 1.5 ml Eppendorf tube with 50 µl DMEM/F12 media. Dissections were performed for up 15 min. The volume of DMEM/F12 meida containing embryos was adjusted to 200 µl  then cells were mechanically dissociated by flicking the tube 30 times and pipetting mixture 10 times through a 200 µl tip. The volume was adjusted to 1 ml with PBS containing 1.0% BSA  and spun to pellet cells at 300 g for 30s. The supernatant was removed and cells were resuspended in 80 µl of PBS containing 0.1% BSA and 20% Optiprep StemCell  Cat 07820  aiming for a concentration above 100 cells/µl. Cells were then passed through a cell sieve 100 µm for dome stage and 70 µm for shield stage. Single cell RNA seq library was performed with Chromium Next GEM Single Cell kit 10x Genomics  Cat PN 1000121 based on standard protocol. All libraries were sequenced by Illumina Hi Seq 1500 or 2500 or XTen platform according to manufacturer’s instruction.,The wild type AB strain was used in the experiments. The females post OMIS were fed with live adult brine shrimp thrice a day. All embryos were raised in Holtfreter’s solution at 28.5C and staged as described previously Kimmel et al.  1995.,strain:AB|genotype:WT|developmental stage:Dome|tissue:embryo|treatment:microinjection with Rhodamine B and control MO at oogenesis III stage GV,GSM5351822,GSM5351822: Dome ctrl scRNA seq rep1; Danio rerio; RNA Seq,GSM5351822,,1,Cell dissociation protocol was based on a previously described method Farrell et al.  2018 with modifications to adapt it for 10x Genomics platform. Briefly  dnmt1 mKD and control embryos were collected 20 min post fertilization and cultured as mentioned before. Then 15 embryos at dome or shield stages for each sample were transferred into plastic Petri dishes that had previously been coated with 2% agarose and soaked with DMEM/F12 media Gibco/Life Technologies  Cat 11330032 at least 2hrs. Next  embryos at desired stages were dechorionated and deyolked manually by forceps and transferred to a 1.5 ml Eppendorf tube with 50 µl DMEM/F12 media. Dissections were performed for up 15 min. The volume of DMEM/F12 meida containing embryos was adjusted to 200 µl  then cells were mechanically dissociated by flicking the tube 30 times and pipetting mixture 10 times through a 200 µl tip. The volume was adjusted to 1 ml with PBS containing 1.0% BSA  and spun to pellet cells at 300 g for 30s. The supernatant was removed and cells were resuspended in 80 µl of PBS containing 0.1% BSA and 20% Optiprep StemCell  Cat 07820  aiming for a concentration above 100 cells/µl. Cells were then passed through a cell sieve 100 µm for dome stage and 70 µm for shield stage. Single cell RNA seq library was performed with Chromium Next GEM Single Cell kit 10x Genomics  Cat PN 1000121 based on standard protocol. All libraries were sequenced by Illumina Hi Seq 1500 or 2500 or XTen platform according to manufacturer's instruction.,GEO Accession:GSM5351822,RNA-Seq,TRANSCRIPTOMIC,cDNA,PAIRED,ILLUMINA,HiSeq X Ten,,SRP322195,,loader:fastq load.py,Dome_ctrl_scRNA_seq_rep1_3_r1.fq.gz Dome_ctrl_scRNA_seq_rep1_3_r2.fq.gz,fastq fastq,10367106900.0,34557023.0,GSM5351822 r3,0:150 1:150,A:2564343754;C:1927723585;G:2020450915;T:3854225526;N:363120,150,150,,,2564343754,1927723585,2020450915,3854225526,363120,SRX11041467,SRS9110397,SRA1239365,GEO,"THU-PKU Center for Life Sciences, Tsinghua University",2,0.33936,0.88676,0.08487,0.08931,0.9513,0.79072,0.58595,0.53954,150,150,T,B,mate1 technical by mapping diff,illumina,hiseq_era,unknown,cdna_unspecified,unknown,sc,single_cell_droplet,10x,,China,2021-06-01,Blastula,Embryo,Embryo Imprecise,All anatomical structures
64446,SRR14703449,SRX11041467,SRS9110397,SRP322195,PRJNA734348,Methylome inheritance and enhancer dememorization construct an epigenetic gate safeguarding embryonic programs,GSE175951,Other,Unlike that of mammals  the total DNA methylome of many cold blooded vertebrates is globally inherited from gametes to early embryos. In zebrafish  this is however accompanied by sweeping “dememorization” of enhancers prior to fertilization for sperm and just post fertilization for oocyte  as they undergo full methylation and are not demethylated again until phylotypic stage. The significance of both global methylome inheritance and enhancer dememorization in early embryos remains largely unknown. Adding to the puzzles  the zygotic mutant zebrafish of dnmt1  the major DNA methylation maintenance methyltransferase  surprisingly can develop to term. To solve the role of DNA methylation in early development  we generated zebrafish embryos derived from dnmt1 knocking down oocytes using a recently developed method OMIS microinjection in situ  which successfully eliminated DNA methylation before zygotic genome activation. dnmt1 deficient embryos failed to initiate epiboly and died around gastrulation. This is in part caused by activation of immune response and p53 regulated apoptosis  likely triggered by the derepression of transposable elements. Single cell RNA seq further revealed defective differentiation in these mutants. DNA methylation is also required for the establishment of repressive histone marks H3K27me3 and H2AK119ub. Strikingly  the loss of DNA methylation leads to extensive derepression of somatic genes and enhancers  which acquire ectopic H3K27ac  accessible chromatin  and H3K4me3. These somatic enhancers are preferentially CG rich and are bound by CG containing TFs. By contrast  embryonic enhancers are generally CG poor  methylation insensitive  and are bound by CG less TFs. Hence  the global DNA methylome inheritance is essential for vertebrate early development  and enhancer dememorization resets an epigenetic gate that separates embryonic and somatic programs. Overall design: By employing MethylC seq  total RNA seq  scRNA seq  CUT&RUN  ChIP seq  and ATAC seq in control and dnmt1 mKD embryos at oocyte  256 cell  dome and shield stages in zebrafish  we interrogated the function of DNA methylome and its inheritance in early zebrafish development.,,pubmed:34936444,,Dome ctrl scRNA seq rep1,GSM5351822,,source name:embryo|strain:AB|genotype:WT|developmental stage:Dome|tissue:embryo|treatment:microinjection with Rhodamine B and control MO at oogenesis III stage GV,Dome ctrl scRNA seq rep1,All STEM seq datasets were mapped to the danRer7 reference genome by Bismark Krueger and Andrews  2011. Reads were trimmed with cutadaptor Martin  2011 using parameters:   minimum length 20   pair filter=any. Alignments were performed with the following parameters:  N 1  X 600   score min L 0  0.6. Multi mapped reads and PCR duplicates were removed. bismark methylation extractor was used to calculate the DNA methylation level. Total RNA seq data were firstly processed using Trim Galore! with default parameters to trim the adapter containing and low quality reads. The filtered data were then mapped to the zebrafish reference genome danRer7 by STAR version: STAR 2.5.3a modified Dobin et al.  2013 with the following parameters:   outSAMstrandField intronMotif   outSAMattributes All   outSAMunmapped Within   outSAMattrIHstart 0   outWigStrand Stranded   outFilterMultimapNmax 20   twopassMode Basic. The gene expression level was normalized to fragments per kilobase of transcript per million mapped FPKM values using Cufflinks version 2.2.1 Trapnell et al.  2012. The scRNA seq data were processed with Cell Ranger 3.1 for genome alignment danRer10  transcript counting  and gene cell barcode matrix generation. Before the downstream analysis  cells with unique detected genes lower than <2 000 and higher than 6 000 genes were discarded  and cells with high percentage of mitochondrial genes >5% were removed too. Subtypes of cells were identified with Seurat 3.0 through the integrating of control and mKD embryos at dome and shield stages with setting control embryos as reference. To make the data comparable samples  we performed SCTransform normalization separately for each dataset. To further reduce the bais caused by sequencing depth of scRNA seq and percentage of mitochondrial genes  UMI variance and percent.mt were regressed out from SCTransform normalized data matrix by linear model with negative binomial distribution. All ChIP seq reads were aligned to the zebrafish reference genome danRer7 using Bowtie2 version 2.2.2 Langmead and Salzberg  2012 with the parameters –t –q –N 1 –L 25. All unmapped reads  non uniquely mapped reads and PCR duplicates were removed. For downstream analysis  the read counts were normalized by computing the numbers of reads per kilobase of bin per million of reads sequenced RPKM. To visualize the STAR ChIP seq signal in the UCSC genome browser  each read was extended by 250 bp and the coverage for each base was counted. Replicates of STAR ChIP seq were pooled for the downstream analysis. The ATAC seq data were mapped to the zebrafish reference genome danRer7 using Bowtie2 version 2.2.2 Langmead and Salzberg  2012 with the parameters –t –q –N 1 –L 25. All unmapped reads  non uniquely mapped reads and PCR duplicates were removed. For downstream analysis  the read counts were normalized by computing the numbers of reads per kilobase of bin per million of reads sequenced RPKM. To minimize the batch and cell type variation  RPKM values across whole genome were further Z score normalized. To visualize the CUT&RUN signals in the UCSC genome browser  each read was extended by 250 bp and the coverage for each base was counted. Genome build: danRer7 Supplementary files format and content: Matrix for scRNA seq data  gene expression table for total RNA seq data  Bigwig and narrowPeak for ChIP seq and ATAC seq data,embryo,Briefly  on the first day of OMIS Wu et al.  2018b  adult females at 5 mpf 12 mpf were anesthetized in 550 µg/ml tricaine Sigma  Cat A5040 in a petri dish. Then the fish was placed on a damp sponge with specific buffer 5.4 mM KCl  136.8 mM NaCl  4.2 mM NaHCO3  0.44 mM KH2PO4  0.25 mM Na2HPO4  and 0.5% wt/vol BSA. A cut was made on one side of belly to expose the ovary. The diluted MOs were microinjected into each oocyte. Rhodamine B Sigma  Cat R8881 was co injected with MOs as a dye. post injection  the wound on the belly was sewed with a surgical sewing needle carefully and quickly. Once the operation was done  the female was transferred into fish water supplemented with 32 µg/ml tricaine  10 unit/ml penicillin and 10 µg/ml streptomycin HyClone  Cat SV30010. Then the fish was transferred to fish water containing gradually reduced concentrations of tricaine. In the evening of the second day  the female was paired with a wild type male. In the morning of the third day  the pair started to chase and lay fertilized eggs naturally. The injected oocyte derived embryos were identified by co injected dye rhodamine B at very early developmental stages. The injection doses of standard control MO cMO were 5 ng per oocyte in the same assay. MOs were dissolved in RNase free water  and heated to 65°C for 10 min before microinjection.,Cell dissociation protocol was based on a previously described method Farrell et al.  2018 with modifications to adapt it for 10x Genomics platform. Briefly  dnmt1 mKD and control embryos were collected 20 min post fertilization and cultured as mentioned before. Then 15 embryos at dome or shield stages for each sample were transferred into plastic Petri dishes that had previously been coated with 2% agarose and soaked with DMEM/F12 media Gibco/Life Technologies  Cat 11330032 at least 2hrs. Next  embryos at desired stages were dechorionated and deyolked manually by forceps and transferred to a 1.5 ml Eppendorf tube with 50 µl DMEM/F12 media. Dissections were performed for up 15 min. The volume of DMEM/F12 meida containing embryos was adjusted to 200 µl  then cells were mechanically dissociated by flicking the tube 30 times and pipetting mixture 10 times through a 200 µl tip. The volume was adjusted to 1 ml with PBS containing 1.0% BSA  and spun to pellet cells at 300 g for 30s. The supernatant was removed and cells were resuspended in 80 µl of PBS containing 0.1% BSA and 20% Optiprep StemCell  Cat 07820  aiming for a concentration above 100 cells/µl. Cells were then passed through a cell sieve 100 µm for dome stage and 70 µm for shield stage. Single cell RNA seq library was performed with Chromium Next GEM Single Cell kit 10x Genomics  Cat PN 1000121 based on standard protocol. All libraries were sequenced by Illumina Hi Seq 1500 or 2500 or XTen platform according to manufacturer’s instruction.,The wild type AB strain was used in the experiments. The females post OMIS were fed with live adult brine shrimp thrice a day. All embryos were raised in Holtfreter’s solution at 28.5C and staged as described previously Kimmel et al.  1995.,strain:AB|genotype:WT|developmental stage:Dome|tissue:embryo|treatment:microinjection with Rhodamine B and control MO at oogenesis III stage GV,GSM5351822,GSM5351822: Dome ctrl scRNA seq rep1; Danio rerio; RNA Seq,GSM5351822,,1,Cell dissociation protocol was based on a previously described method Farrell et al.  2018 with modifications to adapt it for 10x Genomics platform. Briefly  dnmt1 mKD and control embryos were collected 20 min post fertilization and cultured as mentioned before. Then 15 embryos at dome or shield stages for each sample were transferred into plastic Petri dishes that had previously been coated with 2% agarose and soaked with DMEM/F12 media Gibco/Life Technologies  Cat 11330032 at least 2hrs. Next  embryos at desired stages were dechorionated and deyolked manually by forceps and transferred to a 1.5 ml Eppendorf tube with 50 µl DMEM/F12 media. Dissections were performed for up 15 min. The volume of DMEM/F12 meida containing embryos was adjusted to 200 µl  then cells were mechanically dissociated by flicking the tube 30 times and pipetting mixture 10 times through a 200 µl tip. The volume was adjusted to 1 ml with PBS containing 1.0% BSA  and spun to pellet cells at 300 g for 30s. The supernatant was removed and cells were resuspended in 80 µl of PBS containing 0.1% BSA and 20% Optiprep StemCell  Cat 07820  aiming for a concentration above 100 cells/µl. Cells were then passed through a cell sieve 100 µm for dome stage and 70 µm for shield stage. Single cell RNA seq library was performed with Chromium Next GEM Single Cell kit 10x Genomics  Cat PN 1000121 based on standard protocol. All libraries were sequenced by Illumina Hi Seq 1500 or 2500 or XTen platform according to manufacturer's instruction.,GEO Accession:GSM5351822,RNA-Seq,TRANSCRIPTOMIC,cDNA,PAIRED,ILLUMINA,HiSeq X Ten,,SRP322195,,loader:fastq load.py,Dome_ctrl_scRNA_seq_rep1_4_r1.fq.gz Dome_ctrl_scRNA_seq_rep1_4_r2.fq.gz,fastq fastq,11334574200.0,37781914.0,GSM5351822 r4,0:150 1:150,A:2800271980;C:2113897275;G:2209444208;T:4210565270;N:395467,150,150,,,2800271980,2113897275,2209444208,4210565270,395467,SRX11041467,SRS9110397,SRA1239365,GEO,"THU-PKU Center for Life Sciences, Tsinghua University",2,0.34305,0.88505,0.09034,0.08949,0.95264,0.78971,0.60343,0.54776,150,150,T,B,mate1 technical by mapping diff,illumina,hiseq_era,unknown,cdna_unspecified,unknown,sc,single_cell_droplet,10x,,China,2021-06-01,Blastula,Embryo,Embryo Imprecise,All anatomical structures