RNA-seq of Temnothorax rugatulus: queenless workers
The fat bodies from the two workers were collected including the first cuticle plate of the gaster, and pooled in the same Eppendorf tube containing 50 l of TRIzol (Thermofisher) for further RNA sequencing. Tissue collection took less than 10 minutes. The samples were flash-frozen in liquid nitrogen and preserved at -80C. Before the RNA extraction of each sample, we crushed the fat bodies with a pestle, added 50 l of phenol:chloroform:isoamyl alcohol (25:24:1) (Carl Roth), mixed manually, and centrifuged at 1200 xg for 15 minutes. Afterward, the upper phase was transferred to a new tube and mixed with 25 l of ethanol 100% (Carl Roth). RNA was then extracted using a NucleoSpin RNA XS kit (Macherey-Nagel). After quantity and quality control, 20 samples were sent for sequencing (Table 1). Library preparation was conducted following the standard protocol of BGI (Hongkong), which sequenced 150 bp paired-end reads on an Illumina Hiseq X ten.