title: BrmIR37
organism: Drosophila melanogaster
description: Protocols: Stocks used are UAS-mCD8GFP, UAS-Brm-IR (#31712), and Hs-Gal4 from Bloomington Stock Center (http://flystocks.bio.indiana.edu). All transgenes are in the third chromosome. We crossed UAS-Brm-IR males with Hs-Gal4 females to generate the transgenic strain containing UAS-Brm-IR and Hs-Gal4 for Brm knockdown (experimental group, short for BrmIR). Similarly, the transgenic strain containing UAS-mCD8GFP and Hs-Gal4 (control group, short for GFP) was generated by crossing UAS-mCD8GFP males with Hs-Gal4 females and expected to remove the influence of UAS-Gal4 system. Both control and experimental group flies were cultured at normal temperature (25C) for 83.5h (i.e., the third larvae stage) after fertilization. Next, they were cultured at 37C for 30 min to knockdown Brm by the expression of Brm shRNA through heatshock in waterbath, and followed recovery at 25C for 4.5hr. Repeat the heatshock-and-recovery treatment twice, but with recovery time of 3.5hr and 2hr, respectively. Then, the larvae were collected and used in this study. These two groups of flies were denoted as GFP37 and BrmIR37, respectively. Meanwhile, the two groups of flies were cultured at 25C all the time in parallel after fertilization as negative controls. These flies were denoted as GFP25 and BrmIR25, respectively. Total RNA was extracted from 95-99h larvae using TRIzol (Invitrogen). Reverse transcription was performed on 500ng of total RNA in 10uL reaction system for each sample using PrimeScript RT Master Mix Perfect Real Time Kit (TAKARA, Code: DRR036A). The Realtime PCR was carried out in triplicate using F-416L DyNAmoTM ColorFlash SYBR Green qPCR Kit (Thermo). The RNA sequencing libraries were constructed from the extracted RNA using standard Illumina libraries prep protocols.
attributes.tag: ENA-FIRST-PUBLIC
attributes.value: 2014-05-08T17:00:43Z
attributes.tag: ENA-LAST-UPDATE
attributes.value: 2018-03-08T16:49:40Z
attributes.tag: External Id
attributes.value: SAMEA2223466
attributes.tag: INSDC center name
attributes.value: College of Life Science and Technology, Tongji University, Shanghai, China
attributes.tag: INSDC first public
attributes.value: 2014-05-08T17:00:43Z
attributes.tag: INSDC last update
attributes.value: 2018-03-08T16:49:40Z
attributes.tag: INSDC status
attributes.value: public
attributes.tag: Submitter Id
attributes.value: E-MTAB-1966:BrmIR37
attributes.tag: broker name
attributes.value: ArrayExpress
attributes.tag: common name
attributes.value: fruit fly
attributes.tag: developmental stage
attributes.value: larval stage
attributes.tag: genotype
attributes.value: Brm knockdown
attributes.tag: sample name
attributes.value: E-MTAB-1966:BrmIR37
attributes.tag: scientific_name
attributes.value: Drosophila melanogaster
attributes.tag: specimen with known storage state
attributes.value: fresh specimen