GSM5900310: Shal495 1-experimental group; Drosophila melanogaster; RNA-Seq
Adult brains were dissected in phosphate buffer (0.1 M, pH7.2), and fixed for 20 min in 4% paraformaldehyde. RNA was extracted using UNlQ-10 Column Trizol Total RNA Isolation Kit (Sangon Biotech) and treated with DNaseI (BBI) to digest DNA. Oligo dT magnetic beads were used to enrich and purify the qualified RNA samples. Purified mRNA was fragmented and used as a template to synthesize the first strand of cDNA with N6 random primer. The second strand of cDNA was synthesized by adding dNTPs, RNaseH and DNA Polymerase I. Eluted and purified double-stranded cDNA was then repaired with phosphate at 5' end and stickiness 'A' at 3' end, then ligate and adaptor with stickiness 'T' at 3' end. Then the target size fragments are recovered by agarose gel electrophoresis and PCR amplification is performed to complete the entire library preparation. Use Qubit3.0 for preliminary quantification, dilute the library to 1ng/ul, and then use Agilent 2100 to detect the insert size of the library. After the insert size meet expectations, performs QPCR to accurately quantify the effective concentration of the library (>10nM). Sequencing was performed on the prepared library with Illumina platform and paired end (PE) strategy. Sequencing libraries were generated using NEBNext® Ultra™ RNA Library Prep Kit for Illumina® (#E7530L, NEB, USA) following the manufacturer's recommendations and index codes were added to attribute sequences to each sample. 