GSM7329928: Foxj1b Mutant R2; Danio rerio; RNA-Seq
To isolate RNA for sequencing, 4dpf larvae 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 5mins 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 2mins at room temperature and then centrifuged for 15mins 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 30secs at 8000 rpm. The spin column was further incubated with 700μL of RW1 buffer and centrifuged for 30secs 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 45mins at room temperature. After incubation 350μL of RW1 buffer was added to the tubes and centrifuged for 15secs at 8000rpm. The tubes were then treated with 500μL RPE buffer and centrifuged for 30secs. This step was repeated twice, and the tubes were then centrifuged for 1min 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 2mins. The tubes were then centrifuged for 1min at 8000rpm to elute the RNA. The library construction and sequencing was done by BGI's DNBSEQTM Technology.