A multi-valent modified mRNA-LNP vaccine protects against Clostridioides difficile infection
Barcoded PCR primers annealing to the V4 region of the 16S rRNA gene were used for library generation.  PCR reactions were carried out in duplicate using Q5 High-Fidelity DNA Polymerase (NEB, Ipswich, MA).  Each PCR reaction contained 0.5 uM of each primer, 0.34 U Q5 Pol, 1X Buffer, 0.2 mM dNTPs, and 5.0 ul DNA in a total volume of 50 ul.  Cycling conditions were as follows: 1 cycle of 98C for 1 m; 20 cycles of 98C for 10 s, 56 C for 20 S, and 72C for 20 sec; 1 cycle of 72C for 8 m.  After amplification, duplicate PCR reactions were pooled and then purified using a 1:1 volume of SPRI beads.  DNA in each sample was then quantified using PicoGreen and pooled in equal molar amounts.  The resulting library was sequenced on the Illumina MiSeq using 2x250 bp chemistry.