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| Status |
Public on Feb 18, 2016 |
| Title |
Metazome_NV_timecourse_sample_0025 |
| Sample type |
SRA |
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| Source name |
single embryo 25
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| Organism |
Nematostella vectensis |
| Characteristics |
time point: 920
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| Growth protocol |
Eggs and sperms were mixed and egg jelly was dissolved as previously described43 using 4% cysteine (pH 7.4–7.6) to make single embryos accessible for collection. The embryos were washed of cysteine six times using 30% of sea water. Fertilized embryos were observed under light microscope and embryos reaching 4-cell stage were deposited in a 10ul drop of 30% salt water on the cap of a 1.5ml Eppendorf tube. Tubes were closed and incubated for respective periods at 20°C. At collection timings embryos were inspected for viability and excessive water was removed using a micro-mouthpipette. The embryo was then flash-frozen in liquid nitrogen. Stages ranged from 4-cell stage and every 20 minutes for up to 48 hours when embryos reached the late planula stage.
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| Extracted molecule |
total 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. The library was treated with DSN treatment
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2000 |
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| Description |
sample_0025
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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
Read counting with htseq-count version 0.5.4p3.
Genome_build: Nematostella Reference transcriptome Nv.T1 from http://cnidarians.bu.edu/stellabase/assembly/NvT1.fasta
Supplementary_files_format_and_content: Read counting
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| Submission date |
Jul 20, 2015 |
| Last update date |
May 15, 2019 |
| 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 |
GPL17090 |
| Series (2) |
| GSE70185 |
The mid-developmental transition and the evolution of animal body plans |
| GSE71086 |
Nematostella vectensis high resolution developmental transcriptomic time-course |
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| Relations |
| BioSample |
SAMN03890437 |
| SRA |
SRX1100737 |
| Supplementary data files not provided |
SRA Run Selector |
| Processed data are available on Series record |
| Raw data are available in SRA |
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