sample_3b
Total RNA was extracted from the holobiont in fragments stored in TRI-Reagent (Sigma) following the manufacturer's protocol, with some modification at the homogenization step. Briefly, samples frozen in TRI-Reagent were heated and centrifuged, and then bromochloropropane, at a ratio of 1:10, was added to the samples for separation. After incubation at room temperature for 10 minutes and centrifugation, RNA was purified from the clear phase using a Purelink RNA Mini Kit (Ambion) according to the manufacturers protocol. The RNA was washed in 70% ethanol and then on-column DNase digestion was performed using a Qiagen RNase-free DNaseI Kit according to the manufacturer's protocol. RNA-seq libraries were prepared using an in-house protocol at the Weizmann Institute of Science. Briefly, the polyA fraction (mRNA) was purified from 500 ng of total RNA per sample following by fragmentation and generation of double-stranded cDNA. Then, end repair, A base addition, adapter ligation and PCR amplification steps were performed. Libraries were evaluated by Qubit (Thermo Fisher) and TapeStation (Agilent). Sequencing libraries were constructed with barcodes to allow multiplexing of 2 samples in 10 lanes.