INTRO
I was asked to isolate RNA from a list of 60 samples (GitHub Issue) from a number of Crassostrea gigas (Pacific oyster) spat and seed that had been collected as part of the project-gigas-carryover (GitHub repo).
MATERIALS & METHODS
Oysters had been previously preserved in RNAlater (Ambion), supernatant removed, and then frozen @ -80oC. All samples were treated in the following fashion. Here’s the full list of samples (GitHub).
Samples were transferred to 1.5mL SafeLock (Eppendorf) microcentrifuge tubes containig ~100uL of 0.5mm zirconium oxide beads and 250uL of TriZol. Samples were homogenized using the Roberts Lab Bullet Blender 5E Gold + (NextAdvance) with 1.5mL tube adapters for 5mins at speed 12. The Bullet Blender was pre-cooled, and cooled, using dry ice.
Samples with shells which did not appear to fully homogenize were manually disrupted in the existing tube using a disposable microfuge pestle.
After homogenization, 500uL of TriZol was added to each tube, vortexed, incubated at room temperature for ~10mins, and subsequently processed for RNA isolation.
Due to time constraints, the following samples were stored overnight at -20oC:
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