Qiaexcel

Samples were analyzed for amplification of vaccine strain specific gapA sequence via PCR with primers PRUMG32-F/PRUMG36-R [19 (link)] (Table S1). The reaction mix was prepared as reported by Evans and collaborators, and amplification was carried out with a BioRad T100 Thermal Cycler (Bio-Rad, Milan, Italy) according to the published thermal profile. The PCR products were loaded and visualized by capillary electrophoresis with the instrument QIAexcel (QIAGEN, Milan, Italy). The expected size of the vaccine specific gapA amplicon was 110 bp as confirmed by amplification of ts-11 and 6/85.

RILPL2 knockout mouse line was originally acquired from The KOMP Repository and is now available from MMRRC.org (Stock number: 049453-UCD). Rilpl2^{tm1a(KOMP)Wtsi} mice were initially bred with Taconic Total Body Cre mice expressing Cre recombinase (Model 12524) to produce the desired deletion. The mice were then bred and maintained on a C57Bl/6j background to remove the Cre recombinase allele. Genotyping of mice was performed by PCR using genomic DNA isolated from ear biopsies and the following primers, all at 10 pmol/μl: primer 1: 5′-AGTTCCGTGCCCTTTTATAGCTG-3′, primer 2: 5′-ACCACATGGCCTATTACCCAAACT-3′, and primer 3: 5′-ATAATAACCGGGCAGGGGGG-3′. PCR reactions were set up and run using KOD Hot Start Polymerase standard protocol. PCR bands were visualized on Qiaexcel (QIAGEN) using the standard DNA screening kit cartridge. The wild-type sequence leads to a 508-bp species, whereas the knockout produces a 697-bp species. Wild-type, heterozygous, and homozygous RILPL2 knock-out MEFs were isolated from littermate-matched mouse embryos at day E12.5 generated from crosses between RILPL2 knockout heterozygous mice. The genotype of the cell was verified by PCR using genomic DNA as described above.