|Citation||Daines, Bryce, Wang, Liguo, Wang, Hui, Li, Yumei, Emmert, David, Gelbart, William, Li, Wei, Chen, Rui (2010.2.8). Drosophila melanogaster mRNA high-throughput sequencing (Export to RIS)|
|Publication Type||Personal communication to FlyBase|
|Text of Personal Communication||
mRNA-sequencing was applied to determine the transcriptome of Drosophila melanogaster. In total, 272 million paired-end reads in 12 sequencing libraries representing a broad range of Drosophila development were produced.
Collections: all collections were from Canton-S flies.
RNA ID; Stage; Description:
E2-4hr -- early embryos; 0-2hr collection
E14-16hr -- late embryos; 0-2hr collection, sit for 14hrs to get 14-16hr
L3i -- larvae; 3rd instar wandering larvae (~30)
P3d -- pupae; pick 3rd instart larvae into a new vial, collect pupae at 72hr (~30)
FA3d -- adult female; collect virgin into a new vial, collect 48hr later (~30)
MA3d -- adult male; collect male into a new vial, collect 48hr later (~30)
P* -- pupae; pick 3rd instart larvae into a new vial, collect pupae at 1 day
P* -- pupae; pick 3rd instart larvae into a new vial, collect pupae at 2 day
P* -- pupae; pick 3rd instart larvae into a new vial, collect pupae at 3 day
P* -- pupae; pick 3rd instart larvae into a new vial, collect pupae at 4 day
P* -- pupae; pick 3rd instart larvae into a new vial, collect pupae at 5 day
L* -- larvae; lay egg, flip 24 hrs later, collect at 48hr later
L* -- larvae; lay egg, flip 24 hrs later, collect at 72hr later
L* -- larvae; lay egg, flip 24 hrs later, collect at 96hr later
A17d* -- male old; collect new male, age for 17 days
A17d* -- female old; collect new female, age for 17days
E2-16hr -- embryo; lay egg on grape juice plate, flip 14hrs later, age 2 hrs
*Indicates RNA collection was mixed in equimolar amounts for sequencing.
Whole body samples were homogenized in 1ml of Trizol (Invitrogen, Carlsbad, California, USA) per 50-100 mg of tissue using a glass-Teflon homogenizer. Homogenized samples were incubated at room temperature for 5 min. 0.2 ml of chloroform was added and tubes shaken vigorously by hand for 15 seconds followed by incubations at room temperature for 2 to 3 minutes. Samples were then centrifuged at 12,000 x g for 15 minutes at 2 to 8°C. The aqueous phase was transferred to a fresh tube and 0.5 ml of isopropyl alcohol was added. Samples were then incubated at room temperature for 10 minutes and centrifuge at 12,000 x g for 10 minutes at 2 to 8°C. Supernatant was then removed and the RNA pellet washed once with 1ml 75% ethanol followed by a final centrifuge at no more 7,500 x g for 5 minutes at 2 to 8°C. The supernatant was then discarded and the RNA pellet allowed to air dry for 5-10 minutes. Dissolve RNA in RNase-free water.
For each library, mRNA was purified from 20ug aliquot of total RNA using an mRNA Purification Kit (Invitrogen). Double-stranded cDNAs were made from mRNA using the Superscript Double-Stranded cDNA Synthesis Kit (Invitrogen) and random hexamer primers (Invitrogen, 3ug/ul). On a 2% agrose gel, 200-400bp cDNAs were selected and used as the DNA template for the Solexa library construction. The quality and quantity of the resulting double-stranded cDNAs was assessed using the Nanodrop 7500 spectrophotometer (Nanodrop, Wilmington, DE, USA).
Size-selected cDNAs were end-repaired by T4 DNA polymerase and Klenow DNA Polymerase, and T4 polynucleotide kinase. The cDNA products were incubated with Klenow Fragment to generate 3' Adenine overhangs followed by ligation to Illumina adapters. The adapter-ligated products were purified on Qiaquick spin columns (Qiagen), and PCR-amplified with PhusionTM DNA Polymerase using Illumina's genomic DNA primer set. PCR products were purified on Qiaquick MinEluteTM columns and the DNA quality assessed and quantified using a PicoGreen assay and diluted to 10nM. Cluster generation and sequencing was performed on the Illumina cluster station and 1G analyzer following manufacturer's instructions.
Sequencing: all libraries were sequenced on the Solexa/Illumina GAII genome analyzer.
Seq ID; Platform configuration:
E2-4hr -- 65bp barcoded paired-end reads (62bp after removal of barcode)
E14-16hr -- 75bp paired-end reads
E2-16hr -- 75bp paired-end reads
L3i -- 75bp paired-end reads
L -- 75bp paired-end reads
P3d -- 75bp paired-end reads
P -- 75bp paired-end reads
MA3d -- 75bp paired-end reads
FA3d -- 75bp paired-end reads
A17d -- 75bp paired-end reads
E2-16hr100 -- 100bp paired-end reads
L3i100 -- 100bp paired-end reads
Alignment:Reads were aligned to the Drosophila reference genome dm3 using BLAT. The BLAT ooc file was prepared with –repMatch=128 and otherwise default parameters were used. Paired reads were parsed to disambiguate multiple alignment locations if one of the two pairs mapped uniquely or the pair agreed on a unique mapping location. Reads with unaligned mates or pairs which did not agree were treated as single-end reads. Only reads with unique alignment location were considered for analyses.
Total uniquely aligned reads for each sequencing library reported below.
E2-4hr -- 9.0 million
E14-16hr -- 3.3 million
E2-16hr -- 10.1 million
L3i -- 12.6 million
L-- 21.1 million
MA3d -- 5.3 million
FA3d -- 9.6 million
A17d -- 21.4 million
P* -- 22.7 million *This library was sequenced in replicate lanes.
P3d -- 14.6 million
E2-16hr100 -- 6.0 million
L3i100 -- 7.9 million
Total-- 143.6 million
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|Language of Publication||English|
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|Data from Reference|
|Sequence features (54594)|