GSM3374908: nosGAL4>UAS-TKV; Drosophila melanogaster; OTHER
ovaries were dissected in 1XPBS. After dissection, all the PBS was aspirated and 100 μl of Trizol reagent was added to the tissue. The tissue was homogenized. 900 μl of Trizol was added, mixed and incubated at RT for 3 minutes. After incubation, 200 μl of Chloroform was added to each sample and mixed vigorously and incubated at RT for 5 minutes. The samples were then centrifuged at 13,000 rpm for 20 minutes at 4C. The aqueous layer was then transferred to a new centrifuge tube. 2 volumes of 100% ethanol, 10% volume 3 M sodium acetate and 0.5 ul of glycol blue was added to the samples and incubated at -20C for 1 hour. The samples were then centrifuged at 13,000 rpm for 20 minutes at 4C. The pellet was then washed with 75% ethanol, air-dried and re-suspended in RNase free H2O. For efficient re-suspension of the isolated nucleic acid, the sample was incubated at 50C for 10 minutes. The concentration of the isolated RNA was determined using a Nanodrop. 10μg of nucleic acid was then taken and subjected to a DNase treatment using the TURBO DNA-free Kit by Life Technologies (AM1907). poly(A)+ RNA was isolated by double selection with poly-dT beads, using the Turbo DNase treated samples, which is then followed by first- and second-strand synthesis. Sequencing libraries were prepared using NEXTflex Rapid Illumina DNA-Seq Library Prep Kit (Bioo Scientific). Samples were single-end sequenced on a NextSeq 500. Bioo Scientific Manufactures protocol
GEO Accession
GSM3374908