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Status |
Public on Sep 22, 2013 |
Title |
MZpou5f1 Nanog + SoxB1 MO 6hpf poly(A)+ mRNA |
Sample type |
SRA |
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Source name |
MZpou5f1 Nanog + SoxB1 MO 6hpf poly(A)+ mRNA
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Organism |
Danio rerio |
Characteristics |
strain: MZpou5f1 {hi349Tg/hi349Tg} Stage: 6hpf tissue: Whole embryos treatment: Nanog MO, SoxB1 MO
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Treatment protocol |
Treated embryos were injected at 1-cell stage for all treatements except cycloheximide, which was applied by bathing 32-cell embryos and incubating at 27degreesC.
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Extracted molecule |
polyA RNA |
Extraction protocol |
For mRNA-Seq, total RNA from five embryos was extracted using Trizol (Invitrogen) for each experimental condition. RNA was treated with TURBO DNase (Ambion) for 30 minutes at 37°C and extracted using phenol chloroform. For ribosome profiling, 50 wild type embryos for each condition were collected at 64-cell stage. Embryos were lysed using 800ul of a mammalian cell lysis buffer containing 100ug/ml Cycloheximide as per the manufacturer’s instruction (ARTseq Ribosome Profiling Kit, RPHMR12126, Epicentre). For nuclease treatment, 3ul of ARTseq Nuclease was used. Ribosome protected fragments were run and 28-29nt fragments were gel purified as previously described in (Bazzini et al., 2012) and cloned according to the manufacturers protocol (ARTseq Ribosome Profiling Kit, RPHMR12126, Epicentre). TruSeq strand-specific libraries were constructed according to standard protocols. For total mRNA-Seq, sequencing libraries were treated with Epicentre Ribo-Zero Gold kits according to the published protocol, in order to deplete ribosomal RNA prior to sequencing. Ribosome profiling libraries were constructed as in (Bazzini et al., Science 2012).
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Library strategy |
RNA-Seq |
Library source |
transcriptomic |
Library selection |
cDNA |
Instrument model |
Illumina HiSeq 2000 |
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Description |
processed data file: mRNA_exon_rpkm.txt
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Data processing |
Base calling was performed using CASAVA-1.8.2. For mRNA-Seq, raw reads were initially filtered by aligning permissively to a ribosomal DNA index and discarded, using Bowtie v0.12.9 with switches --seedlen 25 -n 3 -k 1 -y -e 10000. Unaligned reads were then aligned strand-specific to the zebrafish Zv9 (UCSC danRer7) genome sequence using Tophat v2.0.7 with default parameters. RPKMs for gene exons are calculated based on joined Ensembl r70 and RefSeq annotations for uniquely aligning reads. Metagenes (with names in the form GeneX.GeneY) are constructed for reads that do not align uniquely to either gene separately, but align to both genes and nowhere else in the genome. The microRNA miR-430 polycistronic precursor is also listed as a meta gene, called ‘mir-430_hairpin’. RPKMs for gene introns (total RNA samples only) are similarly calculated, normalizing against the non-repetitive sum length of a gene’s introns, and the total number of aligned exonic reads in the sample. For Ribosome Profiling, reads were trimmed of 3’ adapter sequence, and reads in the trimmed length range 28-29nts were retained for ribosome protected fragments, >=18nts for input mRNA. Reads were aligned as above, retaining only uniquely aligned reads to the sense CDS sequences of protein-coding genes. Note: traces of exogenous RNA corresponding to a nanog antisense probe, and ntla sense and antisense were detected largely outside the expected size range; these reads were discarded. Gene counts for uniquely aligning reads to CDS regions are listed with respect to RefSeq transcript annotations. Genome_build: Ensembl Zv9 / UCSC danRer7 Supplementary_files_format_and_content: RPKM tables for exons and introns for mRNA-Seq; and RPKM tables for CDS regions for ribosome profiling, generated as described above.
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Submission date |
Aug 29, 2013 |
Last update date |
May 15, 2019 |
Contact name |
Antonio J. Giraldez |
E-mail(s) |
antonio.giraldez@yale.edu
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Phone |
203 785 5423
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Organization name |
Yale University
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Department |
Genetics
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Lab |
Giraldez Lab
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Street address |
333 Cedar Street
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City |
New Haven |
State/province |
CT |
ZIP/Postal code |
06510 |
Country |
USA |
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Platform ID |
GPL14875 |
Series (1) |
GSE47558 |
Nanog, SoxB1 and Pou5f1/Oct4 regulate widespread zygotic gene activation during the maternal-to-zygotic transition |
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Relations |
BioSample |
SAMN02339306 |
SRA |
SRX341252 |
Supplementary data files not provided |
SRA Run Selector |
Raw data are available in SRA |
Processed data are available on Series record |
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