GSM8008066: A. calliptera, female brain, rep1; Astatotilapia calliptera; RNA-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. Quality control, library preparation (directional, with poly-A enrichment), and mRNA sequencing (Illumina, PE150) was performed by Novogene. A. calliptera brain libraries and P.nyererei gonad libraries were prepared differently with the following protocol. We used 50-250 ng of total RNA for library production with the NEBNext® Poly(A) mRNA Magnetic Isolation Module (NEB, #E7490), in conjunction with the NEBNext® Ultra™ II Directional RNA Library Prep Kit for Illumina® (NEB, #E7760) and the NEBNext® Multiplex Oligos for Illumina® (96 Unique Dual Index Primer Pairs, NEB #E6440). Samples were then pooled in equimolar amounts and sequenced on a NovaSeq 6000 system (PE150 on one lane of an S1 Flowcell).