GSM8031705: Smh control, clutch 1; Danio rerio; RNA-Seq
To isolate RNA for sequencing, 30 larvae at 4 dpf were collected in a 1.5ml tube and placed on ice. To lyse the samples, 500μL trizol was added and the euthanized larvae were homogenized through a 27-gauge needle until the mixture looked uniform. After adding another 500μL trizol, the samples were incubated for 5 minutes at room temperature. The larvae were then treated with 200μL chloroform, and the tube was rocked for 15secs to mix the contents. The tubes were incubated for 2 minutes at room temperature and then centrifuged for 15 minutes at 12000rpm at a temperature of 4°C. After centrifugation, the upper aqueous phase containing RNA was mixed with equal amounts of 100% ethanol and was then loaded onto an RNA spin column (Qiagen) and centrifuged for 30 seconds at 8000 rpm. The spin column was further incubated with 700μL of RW1 buffer and centrifuged for 30 seconds at 8000 rpm. The spin column tubes were then placed into a new collection tube and further treated to remove any DNA contamination by washing the tubes with 350μL of RW1 buffer followed by Dnase enzyme (Qiagen) in RDD buffer (10μL Dnase+ 70μL RDD buffer per tube) for 45 minutes at room temperature. After incubation, 350μL of RW1 buffer was added to the tubes and centrifuged for 15 seconds at 8000rpm. The tubes were then treated with 500μL RPE buffer and centrifuged for 30 seconds. This step was repeated twice, and the tubes were then centrifuged for 1 minute at 8000rpm to remove any residual buffer left in the column. For RNA extraction from the column, 30μL nuclease free water was added and incubated for 2 minutes. The tubes were then centrifuged for 1 minute at 8000 rpm to elute the RNA. The concentration of the extracted RNA was quantified using Nanodrop and the quality was analyzed by bioanalyzer. The samples were then sequenced by BGI's DNBSEQTM Technology and bioinformatic analysis was performed by BGI. The filtering of sequencing data was done using SOAPnuke, v1.5.2, using the parameters Parameters -l 15 -q 0.2 -n 0.05. The Hierarchical Indexing for Spliced Alignment of Transcripts software (HISAT2 v2.0.4 with parameters：--sensitive --no-discordant --no-mixed -I 1 -X 1000 -p 8 --rna-strandness RF) was used for mapping RNA-seq reads. We used Bowtie2 (Version：v2.2.5, Parameters：-q --sensitive --dpad 0 --gbar 99999999 --mp 1,1 --np 1 --score-min L,0,-0.1 -p 16 -k 200) to map the clean reads to the reference gene sequence (transcriptome), and then RSEM (Version：v1.2.8, Parameters：-p 8 --forward-prob 0 --paired-end) to calculate the gene expression level of each sample. The count matrix included genes that were selected based on their expression of average count per million of more than 1 across all samples. The resulting matrix was normalized, and log transformed using the voom algorithm from the limma package of Bioconductor (Law et al., 2014; Ritchie et al., 2015). standard procedure performed by BGI