Superscript 3 polymerase

Total RNA was reverse transcribed using SuperScript III polymerase or SuperScript IV VILO Master Mix (Thermo Fisher Scientific) according to the manufacturer’s protocol. For RT-PCR, reactions were performed using the Advantage 2 Polymerase Mix (Clontech) using the primers (Merck) listed in Supplementary file 8. PCR products were gel purified using GeneJET Gel Extraction and DNA Cleanup Micro kits (Thermo Fisher Scientific), cloned into the pGEM T-Easy vector (Promega; according to the manufacturer’s instruction) and sequenced. For RT-qPCR, reactions were carried out using the SYBR Green I (Qiagen) mix with the primers (Merck) listed in Supplementary file 8, following manufacturer’s instructions.

ZIKV-specific primers to generate 5 overlapping amplicons were designed based on an alignment of the reference sequence ZIKV strain MR766 and other complete genomes present in GenBank in February 2016 (Table S1).
ZIKV RNA was isolated from infected cell culture medium using the Qiamp Viral mini kit (Qiagen) and cDNA was generated by reverse transcription (5 separate reactions) with Superscript III polymerase (Thermo Fisher Scientific) and primers A1R, A2R, A3R, A4R and A5R (Table S1) according to the supplier’s instructions. Subsequently, 5 µl cDNA was used in 50 µl PCR reactions with Q5 high-fidelity DNA Polymerase (NEB) with GC enhancer to generate 5 amplicons with sizes of 2.4, 3.3, 3.1, 2.5 and 0.54 kb in 5′ to 3′ order, using primer combinations A1F & A1R, A2F & A2R, A3F & A3R, A4F & A4R and A5F & A5R. An initial step of 30 s at 98 °C was followed by 35 cycles consisting of a 10 s denaturation step at 98 °C, a 30-s annealing step at 64 °C and a 140 s extension at 72 °C for amplicons 1–4, while a 66 °C annealing temperature and 40 s extension time was used to generate amplicon 5. After cleanup with the ISOLATE II PCR Kit (Bioline), DNA was quantified using the Qubit Fluorometer and dsDNA BR Assay (Thermo Fisher Scientific). It was analyzed on agarose gels and the quality was checked with a Bioanalyzer DNA 12000 Kit (Agilent).

The dissected optic nerves were quickly homogenized in lysis buffer, frozen instantly, and stored in liquid nitrogen until further processing. Total RNA was extracted from optic nerves using the Absolutely RNA Nanoprep kit (Agilent Technologies, Santa Clara, CA, USA), then reverse transcribed with Superscript III polymerase (Invitrogen, USA) to synthesize cDNA. Quantitative RT-PCR was performed in the Rotor-Gene Q Cycler (Qiagen) using SYBR GREEN PCR MasterMix (Qiagen) and gene-specific primers (Table 1). For each gene, relative expression was calculated by comparison with a standard curve, following normalization to the expression of housekeeping gene β-actin (Actb; control). We then assessed gene expression 6, and 24 hour’s post SI-TON.Table 1

List of PCR primers.

Gene	Oligonucleotides
Il1b	Forward	GACCTTCCAGGATGAGGACA
	Reverse	AGGCCACAGGTATTTTGTCG
Tnf	Forward	CAAAATTCGAGTGACAAGCCTG
	Reverse	GAGATCCATGCCGTTGGC
Ccl2	Forward	AGGTCCCTGTCATGCTTCTG
	Reverse	ATTTGGTTCCGATCCAGGTT
Cxcl10	Forward	GCTGCAACTGCATCCATATC
	Reverse	CACTGGGTAAAGGGGAGTGA
Gfap	Forward	AGAAAGGTTGAATCGCTGGA
	Reverse	CGGCGATAGTCGTTAGCTTC
Actb	Forward	CACCCTGTGCTGCTCACC
	Reverse	GCACGATTTCCCTCTCAG

Quantitative Real Time PCR qRT-PCR was performed as described [16 (link)]. Briefly, cDNA was generated from 1μg total RNA using oligo-dT primers and Superscript III polymerase (Invitrogen). Real-time PCR analysis was done with iQ SYBR Green Supermix (Bio-Rad) in the Bio-Rad iCycler for 40 cycles. Primer pairs forward (F) and reverse (R): GAPDH F-ATGACAATGAATACGGCTACAG, R-TCTCTTGCTCAGTGTCCTTG; Mylk F-CCAAGGACCGGATGAAGAAATA, R-CCCTGAGATCATTGCCATAGAG; Sema3d F-TGGGACATAGAAGCATTAG, R-AGAGGCTTGTTGGGATTTAGG; Sxc F-AGGGCCTATGAACAGAGAGAT, R-GTAGAGAGCCAGCATGGAAAG; Cadm1 F-TCTGTAGGCGGCTCAGTATAG, R-CTCACATGTCGGGTCTGTTTAG; Krt8 F-GGCCAACCTTAGGAGGAATTT, R-GAGCCAGCTGAGGCTTTATT; Chst7 F-GTGAGACACTGGGACTGATTTG, R-GCCAAGGTGTCTGTCATTACTT; Versican F-CAGGCTATCACAGGCAGATTAG, R-CAGAAGCCAAGGAGTCATTCA.

After seeding THP1-M0 on materials for one day and three days, cells
were detached from the surfaces by pipetting up and down 10 times,
and then cells were transferred into Eppendorf tubes and collected
by centrifuge at 400g for 5 min at room temperature.
The RNA was extracted following the provided protocol from an RNeasy
mini kit (Qiagen), and then cDNA was synthesized using Superscript
III polymerase (Invitrogen). The gene expression of inflammatory cytokines
was measured by real-time PCR (CFX96 Touch, Bio-Rad) and TaqMan probes
for specific cytokine. RPL37 was employed as the
reference gene for the THP-1-derived macrophage.

Library preparation and assembly of the embryonic transcriptomes from E. rowelli were performed as described previously [58] (link). Local tBLASTn searches [73] (link) were conducted using transcriptome libraries from different embryonic stages [58] (link). Previously published sequences from other onychophoran and arthropod species were used as queries [29] (link), [74] (link), [75] (link). RNA was isolated from pooled embryos of different developmental stages using TRIzol Reagent (Invitrogen) and RNeasy MinElute Cleanup Kit (Qiagen, Hilden, Germany) according to the manufacturers’ protocols. First-strand synthesis was performed using random hexamer primers and Superscript III polymerase (Invitrogen). Second-strand synthesis was carried out with DNA Pol I polymerase (Invitrogen). The obtained cDNA was purified using NucleoSpin Extract II-Kit (Macherey-Nagel, Düren, Germany) following the manufacturer's protocol. Fragments of engrailed, cubitus interruptus, wingless and hedgehog were amplified using specific primers (Table 1). The corresponding sequences were made available under the GenBank accession numbers KF218600–KF218603.