RNA extraction and sequencing
Foot and mantel tissues of Coelaturini specimens from Malawi, Uganda and
Zambia were stabilized in RNA-later for subsequent RNA extraction.
Extractions were performed with the NucleoSpin RNA Plus kit of
Machery-Nagel, either according to the protocol of the manufacturer, or
by adding Proteinase K to the lysis buffer. Tissues were disrupted with
magnetic beads (matrix D, 3× 30 s at a speed of 6 ms, MP). The quantity
and quality of mRNA was quantified with high-sensitivity Qubit
fluorometry (Life Technologies Inc.) and with an Agilent BioAnalyzer. We
selected 12 samples from several major clades of Coelaturini based on
the phylogeny of Ortiz-Sepulveda et al. (2020) for sequencing: Six
specimens of Coelatura hypsiprymna and C.
nyassaensis from Lake Malawi Basin; two of Grandidieria burtonifrom Lake Tanganyika; two of Coelatura hauttecoeuri and
two of Nitia acuminata from Lake Victoria (Table S1). RNA
extraction products were purified and poly-A enriched for cDNA
strand-specific, paired-end library preparation with the TruSeq RNA
sample preparation kit v2 (Illumina). The resulting libraries were
sequenced on an Illumina HiSeq 3000 platform v3.0 (2×150 bp).
Purification, library preparation and sequencing were outsourced to UMR
AGAP of the INRA and the University of Montpellier.