alias: GSM2045927
title: GSM2045927: T4T5 replicate2 mRNA-Seq; Drosophila melanogaster; RNA-Seq
library_construction_protocol: The protocol was modified from previously described procedure (Henry et al., Nucleic Acids Res. 2012). 300 µl of Dynabeads Protein G magnetic beads (Thermo Fisher Scientific) were incubated with 10 µl of the monoclonal anti-GFP antibody (Sigma-Aldrich, G6539) for 30 minutes at 4°C. Afterwards, beads were washed three times with 0.1% PBT. Approximately 50 - 60 mL of flies were anesthetized by CO2 and frozen in liquid nitrogen. Flies were decapitated by vigorous vortexing. Heads were smashed using Dounce grinder (Sigma-Aldrich) with loose pestle in ice-cold buffer (10 mM β-glycerophosphate, 2 mM MgCl2, 0.5% Igepal). Homogenate was passed through 180 µm nylon net filter (Millipore) and filtrate was smashed again in Dounce grinder with tight pestle. After additional filtering through 20 µm nylon net filter (Millipore), the homogenate was brought to 50 mL with sucrose buffer (10 mM β-glycerophosphate, 2 mM MgCl2, 25 mM KCl, 250 mM sucrose) and the antibody-preincubated magnetic beads were added. Binding reaction was carried out at 4°C for 30 min and was followed by five washes of the bead-bound nuclei with sucrose buffer. Total RNA from the bead-bound nuclei was extracted with Trizol reagent (Thermo Fisher Scientific). Library was prepared with TruSeq RNA Sample Prep Kit v2 (Illumina)
identifiers.namespace: GEO
identifiers.id: GSM2045927