Project_tenm4-RNA-seq
Description
The flies were placed in 1 mL TRIzol (Invitrogen, USA), and we used forceps to rapidly breakget down the brains of the adult flies rapidly. Almost 20 heads of each genotype were saved in a 1.5 mL microcentrifuge tube with 200 µL of TRIzol. There were 3-4 replicates for each genotype. RNA extraction and purification were performed using TRIzol. The RNA was eluted with nuclease-free water, then a NanoDrop 2000 (Thermo Scientific, USA) was used for theto evaluateion of RNA purity and concentration. RNA-sequencing was performed on All RNA samples, 3-4 replicates of RNA samples from each of elav-GAL4, elav>Ten-m-RNAi, and elav>Ten-m groups. were sent to RNA-sequencing. The sample libraries were prepared according to the standard process of the TruSeq RNA Sample Prep Kit v2 (Illumina, USA). The final libraries were determined by Qubit 2100 (Thermo Scientific, USA) and Bioanalyzer (Illumina, USA) and paired-end sequencing was performed at on an Illumina HiSeq X-Ten System (Illumina, USA), with a read length of 150 2 × 2 150 bp. Adapters and low-quality reads were removed from the raw data using Trimmomatic (v0.36). TopHat (v2.1.1) was used to align the RNA-seq reads to a reference genome (BDGP5.25) with the short-read aligner Bowtie2 (v2.3.4). Cufflinks (v2.2.1) was used to analyze the BAM files, including assembling the transcripts from the aligned reads and estimating their abundances by using a Fragments Per Kilobase of transcript per Million mapped reads (FPKM)-based algorithm. HTSeq (v0.10) was used to generate the read count files for each gene.