Plasmids containing MMR-A or MMR-B genes were used as templates for double-stranded (dsRNA) synthesis.
Thereafter the cDNAs were used as templates for double-stranded cDNA synthesis by PCR amplification using primers that anneal to adapters.
Thereafter, the single-stranded cDNA was used as templates for double-stranded synthesis by PCR amplification using the primer designed according to the adapter sequence.
In brief, we used liquid handling automation to individually select PCR templates for double-stranded RNA (dsRNA) synthesis based on our existing collections.
The ap DG3 chromosome was included because it lacks the DNA corresponding to the homology arms of pLAPGPRA and hence cannot serve as a template for double strand gap repair.
MicroPoly(A Purist kit (Ambion) was used to isolate mRNA from each total RNA pool and 200 ng of mRNA were fragmented and used as template for double stranded cDNA production using cDNA Synthesis System Kit (Roche) followed by adaptor ligation.
Sense and anti-sense catalase-specific primers flanked by the T7 promoter site (Table 1) PCR amplified a 484 bp product from a plasmid obtained from a whole body Lu. longipalpis normalised cDNA library [27] that was used as template for double-stranded RNA synthesis dsRNA.
The cDNA is used as template for double-strand DNA (dsDNA) synthesis and purification.
Total RNA was used as a template for double-stranded cDNA synthesis (Superscript Double-Stranded cDNA synthesis kit, Invitrogen, Carlsbad, CA).
The purified polyA fraction was used as a template for double-stranded cDNA synthesis using the Superscript Plasmid System (Invitrogen Life Sciences, Carlsbad, CA, USA).
The fragmented RNA was then purified using RNAclean XP (Beckman Coulter) magnetic beads and used as template for double-stranded cDNA synthesis using random hexamers (Roche) and a cDNA Synthesis System Kit (Roche) according to the manufacturer's instructions.
