GSM8008048: O. niloticus, ovary, rep3 [smallRNA-seq]; Oreochromis niloticus; ncRNA-Seq
Tissue pieces were transferred to BeadBug tubes prefilled with 0.5 mm Zirconium beads (Merck, #Z763772) and 500-600 µl of TRIzol (Life Technologies, #15596026) was added to the tubes and mixed vigorously. Afterwards, we conducted the homogenisation using a BeadBug microtube homogeniser (Sigma, #Z764140) at approximately 4oC (in cold room). Each sample was homogenised with five BeadBug runs at maximum speed (4000 rpm) for 60 seconds each. Supernatant was then removed into a clean 1.5 mL tube. Centrifuged the lysates again, this time at maximum speed (approximately 21,000 G) for 5 minutes at 4oC. Transferred supernatant into a clean tube without disturbing the pellet and tissue debris. Mixed supernatant thoroughly 1:1 with 100% ethanol, pipetted the mix into a column provided in the Direct-zol RNA Miniprep Plus kit (Zymo Research, #R2072) and followed manufacturer's instructions, using the recommended in-column DNase I treatment. Samples were processed according to the NEXTFLEX® Small RNA-Seq Kit v4 with UDIs (PerkinElmer, #NOVA-5132-32) with a 1 µg starting input and 12 cycles of PCR. Samples were then pooled in equimolar amounts according to the TapeStation results and sequenced on a Novaseq 6000 system (PE50 on one lane of an SP Flowcell).