Qiaminiprep kit

Viral RNA was extracted from SW/GD/2004 and SW/HRB/09 allantoic fluids by using a viral RNA extraction kit (Qiagen, Shanghai, China). Virus-specific cDNAs were obtained by using influenza universal reverse transcription primer uni-12:5′-AGCAAAAGCAGG-3′ with the AMV reverse transcriptase (TaKaRa, Dalian, China). The HA genes were amplified by using HA gene-specific primers, cloned into the pMD18-T vector (TaKaRa, Dalian, China), and then sequenced by using an ABI PRISM 3700 DNA Analyzer (Applied Biosystems, Shanghai Invitrogen, China). Full-length HA was cloned into pCI-neo using specific primers, which introduced a Nhe I/Xho I restriction site. The resulting plasmid pCIneo-HA was purified using a Qiaminiprep kit (Qiagen) as per the manufacturer’s protocols. The extracted plasmid was identified by using a double digest of Nhe I and Xho I (New England Biolabs, Whitby, ON, Canada). Recombinant plasmids were transformed into TOP10 competent cells. Colonies were screened via PCR to confirm insertion of the gene segments. The plasmid sequencing was conducted by using an ABI 3730 DNA automatic sequencer.

ryfA1 was amplified from the S. dysenteriae chromosome [15 (link)] using specific primers containing MfeI and HindIII restriction sites (Table S1). ryfB1 was also amplified from the S. dysenteriae chromosome using primers containing MfeI and SacI restriction sites. Amplicons were run on an agarose gel and purified using the QIAquick gel extraction kit (QIAGEN) as per the protocol. Restriction enzymes MfeI and HindIII from (New England Biolabs Inc., Ipswich, MA, USA) were used to digest the ryfA1 containing amplicons as well as plasmid pQE2, an expression plasmid with an isopropyl β-D-1-thiogalactopyranoside (IPTG) inducible promoter (QIAGEN). Restriction enzymes MfeI and SacI from (New England Biolabs Inc.) were used to digest the ryfB1 amplicon as well as pQE2. Both ryfA1 and ryfB1 were subsequently ligated into pQE2 (QIAGEN) using T4 ligase (New England Biolabs Inc.) creating plasmids pRyfA1 and pRyfB1 respectively (Table S2). The resulting plasmids were introduced into competent E. coli K12 DH5α using heat shock transformation. Each plasmid was then extracted from DH5α using a QIAmini prep kit (QIAGEN) as per their instructions, sequence verified, and introduced into competent S. dysenteriae using electroporation.

Acr-Hox gene-specific primer design was performed with the sequence assembling software Geneious 6.1.6 (Biomatters Limited) and primers were purchased from Life Technologies Company (Thermo Fischer Scientific). First strand cDNA was synthesized by reverse transcription of RNA pooled from representative developmental stages covering the entire larval and early post-metamorphic development (cDNA synthesis kit, #04379012001, Roche Diagnostics). Hox gene sequences were amplified with the gene-specific primers via standard PCR. PCR products were cloned by insertion into pGEM-T easy vectors (#A1360, Promega) and plasmid minipreps were purified with Qia-miniprep kit (#27106, Qiagen). Antisense and sense probes from linearized plasmid sequences were synthesized with a DIG-labeling kit (#11277073910, Roche Diagnostics).