Analyzing the effect of hematopoietic LCN2 on the microbiome in GVHD
Variable (V) region 3 and 4 amplicons of 16S rRNA gene were sequenced following 16S sequencing library protocol by Illumina. 16S Amplicon PCR Forward Primer 5 TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGCCTACGGGNGGCWGCAG and 16S Amplicon PCR Reverse Primer 5 GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG GACTACHVGGGTATCTAATCC were chosen as specific primers for the regions of interest of the 16 S rRNA gene (V3 and V4). Libraries were prepared by performing Amplicon PCR, product cleanup, Index PCR and second product cleanup according to protocol provided by Illumina. Quantification and quality was assessed by loading on a TapeStation (Agilent). Library was denatured with NaOH, diluted, mixed with 20% PhiX control and finally loaded on MiSeq for high-throughput sequencing (2 x 300 cycle V3 kit).