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| Status |
Public on Feb 18, 2016 |
| Title |
Metazome_Drosophila_timecourse_sample_0064 |
| Sample type |
SRA |
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| Source name |
single embryo 64
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| Organism |
Drosophila melanogaster |
| Characteristics |
time (minutes cellularization stage): 930
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| Growth protocol |
Drosophila melanogaster embryos were collected using the protocol published in (PMID:21357063). In short, agar plates with apple juice smeared with freshly prepared yeast were used to make young adult flies lay a lot of eggs. Cages consisting of such plates were set up with at least 20 flies and left for roughly one day for the flies to acclimatize to the cage. In order to make sure to use only newly laid eggs the apple juice plates were replaced with fresh ones twice in one hour interval in the morning to clear the embryos held by the mothers overnight as fresh food stimulates egg laying. The third apple juice plate contained embryos that were further processed for embryo collection. Drosophila embryos are covered with a non-transparent chorion which has to be removed prior to live imaging by dechorionation. Shortly after being laid embryos were washed off from plates into a plastic sieve using tap water and a fine brush to loosen the embryos. In the sieve embryos were submerged into 50% bleach solution for exactly two minutes. Embryos were washed off from bleach on the sieve with cold tap water by spraying with a plastic squirt. Embryos were finally rinses by dipping the plastic sieve three times into white plastic weighing baskets filled with fresh water and then left in the last basket until further handling. Using a needle pick 20 embryos were placed in a row on a strip of agar placed on a glass slide. n-Heptane glue was applied in a thin layer on a big glass cover slip. This cover slip was carefully put upside down on top of the embryos on the agar strip so that embryos adhere to the glue layer. Embryos were covered with PBS and kept in humid chambers in a 25°C incubator. Embryos on the slip were observed under light microscope and for each embryo time of cellularization was noted. Reaching respective time after cellularization embryos were carefully removed from slide using needle pick and flash frozen in an Eppendorf tube in liquid nitrogen.
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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.
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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 BDGP5
Read counting with htseq-count version 0.5.4p3. Using on Ensemble BDGP5.76 annotations
Genome_build: BDGP5
Supplementary_files_format_and_content: DM_exp.tab is a tabular Expression matrix reporting read counts.
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| Submission date |
Aug 18, 2014 |
| 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 |
GPL13304 |
| Series (2) |
| GSE60471 |
Drosophila melanogaster high resolution developmental transcriptomic time-course |
| GSE70185 |
The mid-developmental transition and the evolution of animal body plans |
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| Relations |
| Reanalyzed by |
GSM3286656 |
| BioSample |
SAMN02996785 |
| SRA |
SRX682340 |
| 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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