title: Patient ID:HC2_HC4, corisin amplicon
design: Two sequences flanking the proapoptotic peptides (SVKAQF and WGTGSV) are highly conserved, and the conserved codon usage in the genes allowed the design of two primers, Corisin-F 5ATCAGTTAAAGCTCAATTC and Corisin-R 5GCTACTGAACCAGTACCCCATG, as the forward and reverse primers, respectively. The primer pair amplifies ~150bp DNA fragments containing the coding sequences of the proapoptotic peptides. The primers were validated by extracting community genomic DNA from the urine of healthy controls (n=4) and patients with diabetic CKD (n=18). The right size fragment was amplified from all extracted genomic DNA, except for the negative control (same PCR mixture, without DNA), and the corisin/corisin-like peptides in the urine samples were obtained by using the DNA sequencing and following bioinformatics analyses. Each PCR product was resolved in agarose gel and purified (QIAquick PCR purification kit, Qiagen). Each PCR product was considered a library of the kidneys and urinary tract of a particular CKD patient or healthy control. The DNA was next used in preparing unique libraries by using the NEBNext DNA Library Prep Master Mix Set for Illumina with Unique Dual Indexes to prevent index switching. Library preparations was done using an EpMotion 5075 liquid handler (Eppendorf). The individually barcoded libraries were amplified, quantitated with Qubit and resolved on a Fragment Analyzer to confirm the absence of free primers and primer dimers and the presence of DNA of the expected size. Libraries were pooled in equimolar concentration and further quantitated by qPCR on a BioRad CFX Connect Real-Time System (Bio-Rad Lab Inc., CA). The pooled barcoded shotgun libraries were loaded on a MiSeq flowcell for sequencing (Illumina).