characteristics.text: ERC000011
characteristics.characteristic: ENA-CHECKLIST
characteristics.text: 2015-06-17T17:01:16Z
characteristics.characteristic: ENA-FIRST-PUBLIC
characteristics.text: 2018-03-08T20:53:30Z
characteristics.characteristic: ENA-LAST-UPDATE
characteristics.text: SAMEA2743234
characteristics.characteristic: External Id
characteristics.text: The Pirbright Institute
characteristics.characteristic: INSDC center name
characteristics.text: 2015-06-17T17:01:16Z
characteristics.characteristic: INSDC first public
characteristics.text: 2018-03-08T20:53:30Z
characteristics.characteristic: INSDC last update
characteristics.text: public
characteristics.characteristic: INSDC status
characteristics.text: ERS538975
characteristics.characteristic: SRA accession
characteristics.text: E-MTAB-2880:L3_12h_M
characteristics.characteristic: Submitter Id
characteristics.text: 12 hour
characteristics.ontologyTerms: http://purl.obolibrary.org/obo/UO_0000032
characteristics.characteristic: age
characteristics.text: ArrayExpress
characteristics.ontologyTerms: http://www.ebi.ac.uk/efo/EFO_0009737
characteristics.characteristic: broker name
characteristics.text: African malaria mosquito
characteristics.characteristic: common name
characteristics.text: 45-50 3rd instar larvae collected 12-14 h after ecdysis constituted the sample Protocols: Three days post-blood feeding females were allowed to lay eggs for 10-15 min, after which the egg dish was removed and kept at standard insectary conditions (27 C and 12h light/12h dark cycle). Individual embryos were sexed by PCR using a combination of primers targeting the Y chromosome-specific and the non-Y chromosome repetitive markers (Krzywinski et al. 2004, Genetics 166: 1291; Krzywinski et al. 2005, Genetics 169: 185). Larvae were reared at low density in pans with de-ionized water and fed daily with ground Tetramin fish food. Larvae were collected from progeny of a cross between Pimperena strain females and males of a transgenic strain with an X-linked GFP/dsRED marker on a Pimperena genetic background. Pans containing fully grown 2nd or 3rd instar larvae were inspected every two hours, and newly moulted 3rd or 4th instar individuals were transferred into marked pans for subsequent sample collection. Similarly, pans with fully grown 4th instar larvae were inspected every hour and newly pupated individuals were transferred into marked pans with clean de-ionized water. Transferred individuals were sexed and then collected at required time intervals. Larvae were sexed under a fluorescence microscope based on the principle of inheritance of the fluorescently labelled X chromosome by F1 females from transgenic fathers and lack of fluorescent markers in F1 males. Pupae were sexed based on morphological characters. Individual embryo samples of the same sex (determined by PCR) were combined prior to RNA extraction. Total RNA was isolated using PureLink RNA Mini Kit (Ambion) with an on-column DNase treatment step according to the manufacturer's protocol. Ribo-Zero, Magnetic Gold Kit was used to deplete rRNAs from total RNA prior to library construction. The libraries were constructed using a modified TruSeq RNA protocol (Illumina). The total RNA was fragmented and the first-strand cDNA synthesized. During the second-strand synthesis, dDUTP was incorporated instead of dTTP. The double-stranded cDNA fragments were blunt-ended and their 3-prime ends adenylated to prevent formation of chimeric molecules during the adapter ligation. After adapter ligation, the fragments were treated with Uracil-N-Glycosylase to selectively degrade the second-strand of cDNA. Then, the fragments were subjected to a bead-based size selection using AMPure XP beads. Prior to hybridisation to the flow cell, the cDNA constructs were amplified by PCR to selectively enrich fragments with adapter molecules on both ends. The concentration of the libraries was determined by using a Qubit dsDNA HS Assay (Life Technologies).
characteristics.characteristic: description
characteristics.text: 3rd instar larvae
characteristics.ontologyTerms: http://www.ebi.ac.uk/efo/EFO_0002682
characteristics.characteristic: developmental stage
characteristics.text: Anopheles gambiae
characteristics.characteristic: organism
characteristics.text: Anopheles gambiae
characteristics.ontologyTerms: http://purl.obolibrary.org/obo/NCBITaxon_7165
characteristics.characteristic: organism
characteristics.text: Anopheles gambiae
characteristics.characteristic: scientific_name
characteristics.text: male
characteristics.ontologyTerms: http://purl.obolibrary.org/obo/PATO_0000384
characteristics.characteristic: sex
characteristics.text: fresh specimen
characteristics.ontologyTerms: http://purl.obolibrary.org/obo/OBI_0000971
characteristics.characteristic: specimen with known storage state
characteristics.text: Pimperena
characteristics.characteristic: strain
characteristics.text: L3_12h_M
characteristics.characteristic: title