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| Status |
Public on Feb 18, 2016 |
| Title |
Metazome_Tardigrade_timecourse_sample_0084 |
| Sample type |
SRA |
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| Source name |
single embryo 84
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| Organism |
Hypsibius dujardini |
| Characteristics |
time (minutes after fertilization): 240
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| Growth protocol |
Hypsibius dujardini starting cultures were provided by Bob Goldstein (University of North Carolina at Chapel Hill) and were grown as previously described (Gabriel et al. 2007). Small cultures of tardigrades were kept in 60 mm glass Petri dishes in commercial bottled spring water until gravid animals were visible. Tardigrades lay 2-5 eggs during molting, with the embryos deposited in their exuvia, the shed exoskeleton. Soon after the adult crawled out of the exuvia, it was cut open using a scalpel on a microscope-cavity-slide to release embryos into the medium. Embryos were observed using a standard binocular and when reaching two-cell stage were deposited in a 10 ul drop mineral water on the cap of an 1.5 ml Eppendorf tube. Tubes were closed and incubated for respective periods at 20°C. For 4.5 days every hour past two-cell stage embryos were inspected for viability and excessive water was removed using a micro-mouth pipette. The embryo was then quick-frozen in liquid nitrogen.
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| Extracted molecule |
polyA RNA |
| Extraction protocol |
Total RNA was extracted from single embryos using trizol as described in (Levin et al. Developmental cell 2012) including minor adjustments. After the addition of trizol to the embryos the mixture was frozen in liquid nitrogen, thawed at 37 degrees and vortexed for 30 seconds. This procedure was repeated five times. Only then chloroform was added and the sample further processed. Dried total RNA pellet was dissolved in RNAse free water before introduction into subsequent amplification and sequencing library preparation steps.
The CEL-Seq protocol (Hashimshony, et al. Cell reports 2012) was used to amplify and sequence RNA from whole embryos. CEL-seq multiplexing barocdes were used.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2000 |
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| Data processing |
Libraries were sequenced on the Illumina HiSeq2000 according to standard, paired-end sequencing, protocols. Primary analysis done in RTA 1.17.20,1.13.48
Conversion from BCL to FASTQ and deumltiplexing using CASAVA-1.8 (configureBclToFastq.pl--fastq-cluster-count 1234567890 --mismatches 0 --use-bases-mask Y15n,I6n,Y35n)
Filter and read trimming (barcode minimum quality of 10. trimming of read2 to 35 bases - not required).
CEL-Seq (Hashimshony, et a. 2012l) demultiplexing of second mate, using first mate barcode, allowing no mismatches in barcode.
bowtie2, version 2.1.0, against Hduj_transcriptome_v1
Read counting with htseq-count version 0.5.4p3.
Genome_build: Hduj_transcriptome_v1 (NCBI BioProject PRJNA271450)
Supplementary_files_format_and_content: tabular expression matrix
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| Submission date |
Jan 13, 2015 |
| Last update date |
Jun 12, 2020 |
| Contact name |
Itai Yanai |
| E-mail(s) |
yanai@technion.ac.il
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| Organization name |
Technion - Israel Institute of Technology
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| Department |
Biology
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| Lab |
Yanai
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| Street address |
Technion City
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| City |
Haifa |
| ZIP/Postal code |
30200 |
| Country |
Israel |
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| Platform ID |
GPL19649 |
| Series (2) |
| GSE64944 |
Hypsibius dujardini high resolution developmental transcriptomic time-course |
| GSE70185 |
The mid-developmental transition and the evolution of animal body plans |
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| Relations |
| BioSample |
SAMN03283605 |
| SRA |
SRX840803 |
| 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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