Project_tenm4-RNA-seq

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.将果蝇置于1 mL TRIzol试剂（Invitrogen，美国）中，随后用镊子快速解离成年果蝇的脑组织。每种基因型的果蝇取近20头头部，置于含200 µL TRIzol的1.5 mL微量离心管中，每种基因型设置3-4次生物学重复。采用TRIzol法完成RNA的提取与纯化，使用无核酸酶水洗脱RNA，随后利用NanoDrop 2000超微量分光光度计（赛默飞世尔科技，美国）检测RNA的纯度与浓度。 对所有RNA样本开展RNA测序（RNA-seq），其中包含elav-GAL4、elav>Ten-m-RNAi及elav>Ten-m三组样本各3-4次生物学重复的RNA样品。参照TruSeq RNA样本制备试剂盒v2（Illumina，美国）的标准流程构建测序文库。使用Qubit 2100荧光定量仪（赛默飞世尔科技，美国）与生物分析仪（Illumina，美国）对最终文库进行质检，随后在Illumina HiSeq X-Ten测序平台（Illumina，美国）上完成双端测序，测序读长为2×150 bp。 使用Trimmomatic（v0.36）对原始测序数据进行接头序列及低质量读段（reads）的过滤。借助短读长比对工具Bowtie2（v2.3.4），通过TopHat（v2.1.1）将RNA-seq读段（reads）比对至参考基因组BDGP5.25。使用Cufflinks（v2.2.1）分析BAM格式文件，包括从比对后的读段（reads）中重构转录本，并基于每百万映射读段每千碱基转录本片段数（Fragments Per Kilobase of transcript per Million mapped reads, FPKM）的算法估算转录本丰度。采用HTSeq（v0.10）为每个基因生成读段计数文件。