GSM2053226: Sample_f_48_SuTpl_rep2; Drosophila melanogaster; RNA-Seq
Flies were removed from the indicated incubation temperatures and dissected immediately in Phosphate Buffered Saline solution at room temperature. The testes were removed at the junction with the vas deferens.  Ovaries were removed at the common oviduct, and any eggs in the ovipositor were also removed.  Each dissected fly was immediately crushed in 100ul RNAlater solution  (Life Technologies, Carlsbad, CA) and sealed in a deep-well 96-well plate at room temperature after dissection and during transportation. After the samples arrived at NIH they were stored at -80oC. For RNA extraction, 20-30 glass beads (1.0mm dia. GLASS BEADS Cat. No. 11079110, BioSpec Products, Bartlesville, OK) were added to each well of samples with 100ul frozen RNAlater. Then we added 600ul RLT buffer from the RNeasy 96 kit (Qiagen, Valencia, CA) to each well. After all samples were completed thawed, we homogenized the samples three times for 60 seconds each with a mini-beadbeater (Biospec Products, Bartlesville, OK). Extraction of total RNA was performed using the RNeasy 96 kit according to manufacturer's guide (RNeasy 96 Protocol for Isolation of Total RNA from Animal Cells, III Using spin technology centrifuge), except that we used 700 μl of 70% ethanol in each well to dilute the RNAlater and RLT buffer. We mixed 400ng of total RNA in 50ul of nuclease-free water (Ultra Pure, Quality Biological, Gaithersburg, MD). Then we followed a strand-specific library preparation pipeline (Lee at al., 2015, doi: http://dx.doi.org/10.1101/032920). Libraries were quantified with Quant-iT PicoGreen (Life Technologies, Carlsbad, CA), and pooled for multiplexed sequencing.
GEO Accession
GSM2053226