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Sample GSM1480625 Query DataSets for GSM1480625
Status Public on Feb 18, 2016
Title Metazome_Drosophila_timecourse_sample_0011
Sample type SRA
 
Source name single embryo 11
Organism Drosophila melanogaster
Characteristics time (minutes cellularization stage): 135
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.
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.
 
Library strategy RNA-Seq
Library source transcriptomic
Library selection cDNA
Instrument model Illumina HiSeq 2000
 
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.
 
Submission date Aug 18, 2014
Last update date May 15, 2019
Contact name Itai Yanai
E-mail(s) yanai@technion.ac.il
Organization name Technion - Israel Institute of Technology
Department Biology
Lab Yanai
Street address Technion City
City Haifa
ZIP/Postal code 30200
Country Israel
 
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
Relations
Reanalyzed by GSM3286603
BioSample SAMN02996731
SRA SRX682287

Supplementary data files not provided
SRA Run SelectorHelp
Raw data are available in SRA
Processed data are available on Series record

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