GSM8896147: Hemocyte, adult; Apis mellifera; RNA-Seq
RNA was extracted from hemocytes from 320 µL of hemolymph, equivalent to approximately 5 million hemocytes. The hemolymph was centrifuged at 0.5G for 5 minutes at 4°C and the plasma was discarded. RNA extraction from the resulting hemocyte pellets was carried out using the Qiagen RNeasy Micro Kit. The pellet was resuspended in 350 µl of RLT lysis buffer supplemented with 1% β-mercapto-ethanol and vortexed. The lysate was then passed through a 20G needle ten times to ensure thorough disruption of the hemocytes. The RNA was purified using the Qiagen RNeasy spin column following the manufacturer's recommendations, including an on-column DNase step. Finally, the RNA was eluted in 14 µL of RNase-free water. RNA sequencing was performed by BGI Genomics (China). mRNA enrichment was conducted using oligo dT beads to target mRNA molecules with poly A tails. In the next step, mRNA was fragmented into smaller pieces and then converted into first-strand cDNA using random primers. The second-strand cDNA synthesis utilized dUTP. The resulting cDNA underwent end-repair and 3' adenylation, followed by adapter ligation to the 3' adenylated cDNA fragments. PCR amplification was employed for library fragment enrichment, followed by quality control and library circularization steps. Subsequently, the library underwent amplification to generate DNA nanoballs, enabling sequencing on the DNBSEQ platform with paired-end reads of 150 base pairs.