Total RNA isolation, cDNA synthesis, cloning, sequencing, subcloning and expression in E. coli

The aliform parapodia tissue was excised from the worm (Fig. 1A) and stored in RNA Later solution at 4 °C until the extraction. The total RNA was isolated using TRIzol Reagent and PureLink RNA isolation kit (Invitrogen) according to the manufacturer’s instructions. The first strand cDNA was synthesized from 320 ng total RNA using 18mer oligo-dT (IDT) and M-MLV RT. A full-length cDNA was amplified by PCR using Native Taq DNA polymerase (Invitrogen) and primers 5′-ATG GCC CAG ACH CAG CCN CG-3′ (forward) and 5′-TTA GCT GCT CAG GCT CTC CTT-3′ (reverse). The PCR was programmed as follow: initial denaturation at 95 °C for 2 min, then 35 cycles of 94 °C for 40 s, cooling at 46.4 °C for 1 min, and 72 °C for 1 min followed by a final extension at 72 °C for 10 min. A 519 bp PCR fragment was purified from agarose gel using Zymoclean Gel DNA Recovery kit (Zymo Research) and cloned into PCR-4 TOPO vector (Invitrogen) and transformed in One Shot TOP-10 competent cells (Invitrogen). The construct was verified by DNA sequencing. The cloned DNA insert was PCR amplified using high fidelity Pfx polymerase (Invitrogen) and primers 5′-CACC ATG GCC CAG ACT CAG CCG CG-3′ (forward) and 5′-TTA GCT GCT CAG GCT CTC CTT-3′ (reverse) PCR program 94 °C for 3 min then 30 cycles of 94 °C for 15 s, 60 °C for 30 s, 68 °C for 1 min followed by a final extension at 68 °C for 10 min. The amplified DNA, which showed best match to a ferritin-like protein, was gel purified and sub-cloned into pET 200 Directional TOPO expression vector (Invitrogen). The recombinant plasmid carrying ferritin-like cDNA was then transformed into BL21 star (DE3) E. coli cells (Invitrogen) to produce recombinant protein, worm ferritin (ChF). E. coli cells carrying recombinant were cultured overnight (16 h) at 37 °C in 20 mL LB containing 50 μg/mL kanamycin with shaking at 210 rpm. The overnight grown culture was transferred to a fresh 500 mL LB containing kanamycin and was grown until A_600 nm reached 0.7–0.8. To induce expression, Isopropyl β-D-thiogalactopyranoside was added at a final concentration of 1 mM. The culture was further incubated at 16 °C overnight under agitation at 250 rpm. The induced bacterial cells were pelleted down by centrifugation at 2,000 g for 20 min at 4 °C. The pellets were then stored at −80 °C until use.