title: PGD.human.metagenome
design: RNA was extracted from collected supernatant using the Qiagen miRNeasy Mini Kit (Qiagen, Hilden, Germany Cat#74135). Quality control was established with RNA integrity number (RIN) cut-off >6. RNA sequencing was performed using HiSeq (Illumina, San Diego, CA) at the NYU Genomic Technology Center. RNA-Seq library preps were made using Illumina TruSeq Stranded mRNA LT kit (Illumina, San Diego, CA Cat#RS-1222-2101) on a Beckman Biomek FX instrument, using 250 ng of total RNA as input, amplified by 12 cycles of PCR, and run on an Illumina 2500 (v4 chemistry), as single-read 50bp. Sequences from the murine lung homogenate were aligned against the murine ensemble reference genome utilizing STAR (v2.5) aligner (61). Gene counting of each sample was performed using featureCounts (v1.5.3) (62, 63) and gene differential expression was calculated using the DESeq2 (v3.5) package on R (v3.6) (64). Genes with an adjusted p-value (FDR) q<0.10 were considered significantly differentiated, unless otherwise specified. Ingenuity Pathway Analysis (IPA) was used to identify the most differential biological functions, disease/disorders, regulator effect networks, and to generate top canonical/network pathways (Qiagen, Hilden, Germany https://qiagenbioinformatics.com/products/ingenuity-pathways-analysis)