GSM9686002: aipwh86(+/+)_1; Danio rerio; RNA-Seq
A Thermo Fisher KingFisher Apex liquid handler system and a Zymo Quick-RNA MagBead kit were used to perform the extractions in a 96-well format. The Zymo kit requires an equal volume of ethanol to be mixed with the sample for extraction but has a max volume of 1000 µL. Since 550 µL of supernatant was collected, the final volume with ethanol was 1100 µL and had to be split between two extraction plates. Each sample was individually moved and equally divided between two extraction plates. The well position of the original sample plate was tracked throughout this process and into the sample submission. Ethanol and mag beads were added using a multichannel pipette and the extraction was carried out following the kit protocol including a DNase I digestion step. The RNA was eluted into 50 µL of UP water. Individual larval RNA samples were pooled following RNA extraction to reduce inter-replicate variation. Each pooled replicate was created by combining five individual samples. Samples with the highest concentration and RIN scores > 8 were selected to be pooled. The RIN and concentration of each sample was determined using an Agilent 4150 TapeStation System and an Invitrogen qubit fluorometer, respectively. Library preparation was performed using the CORALL mRNA-Seq V2 whole transcriptome sequencing kit. 150bp paired end reads were sequenced on an AVITI sequencing platform