Barseq analysis of 25 different SC5314 strains from different laboratories in murine model of colonization as well as systemic infection
The Bar-seq libraries were custom made by a protocol developed in the laboratory and were prepared from gDNA isolated from either fecal pellets, GI or systemic organs using the Zymobiomics DNA Miniprep kit (Zymo Research). The one step preparation of Bar-seq libraries was carried out using different combinations of i5 and i7 indices containing primers for multiplexing. 1-3 l of undiluted gDNA from fecal pellets or organs was used per 20 l of PCR reaction. The PCR was carried out using Phusion polymerase and DMSO was used additionally. The following PCR conditions were utilized: 95C for 5 min, 95C for 20 sec, 60C for 20 sec, 72C for 20 sec, steps 2-4 repeated for 34 cycles, 72C for 5 min. All PCR reactions resulted in an amplicon of 250 bp. The multiple PCR samples from the same gDNA were pooled together and resolved on 2% agarose gel which was followed by gel purification to generate final Bar-seq libraries. The concentration of different libraries was determined by Nanodrop and qPCR and sizes were confirmed on a bioanalyzer.