GSM6754839: ko_am30_2; Danio rerio; RNA-Seq
At -3, 0, and 2.5 hours zeitgeber time (onset of the light phase in the incubator) on day 5 post-fertilization, four biological replicates of klf9-/- mutant and wildtype larvae were collected as follows. Each biological replicate from each genotype and timepoint consisted of six larvae in a 30mm dish in 4ml of media. Larvae were collected from the dish into Eppendorf tubes, media was removed, and larvae immediately snap frozen in liquid nitrogen. Collection of the 8 samples at each time point occurred over ~10 minutes. Frozen samples were stored at -80 deg C. RNA was prepared using the Qiagen RNeasy Plus mini kit (Qiagen 74134). For each set of four biological replicate samples, RLT Plus lysis buffer (plus beta-mercaptoethanol, per kit instructions) was added to each sample immediately upon its retrieval from the freezer, and the sample was then immediately homogenized with a motorized pestle and placed on ice before the next sample was retrieved from the freezer and subjected to the same procedure. Following pestle homogenization replicate samples received simultaneous processing—i.e., passed though Qiashredder columns (Qiagen 79654), bound, washed, and eluted per kit instructions. RNA was quantified and sent to the Oklahoma State Genomics Facility for Illumina library preparation and single-end sequencing.