Quant it picogreen assay

Fecal boli samples were collected from CON and DEP+MS dams at E17.5 (n = 14 dams total). Bacterial taxa were identified using 16S rRNA sequencing of microbiome samples. Library preparation was conducted in accordance with standard protocols (earthmicrobiome.org). First, DNA was extracted from all samples using a DNeasy Powersoil Kit (Qiagen, Germantown, MD). Next, PCR with individually barcoded primers (515F-806R; (Apprill et al., 2015 ; Caporaso et al., 2012 (link); Caporaso et al., 2011 (link); Parada et al., 2016 (link)) was used to amplify the V4 hypervariable region of the 16S rRNA gene. PCR product was then purified (PCR Purification Kit, Qiagen, Germantown, MD), DNA concentration was measured using a Quant-iT Picogreen Assay (Thermofisher Scientific), and an equimolar pool of all samples was made and transferred to the core Duke Microbiome Core Facility for sequencing on an Illumina MiSeq Sequencer (Illumina, San Diego, CA, USA).
The Qiime2–2019.7 analysis platform was used to analyze 16S data. Briefly, forward and reverse reads were imported, demultiplexed, and quality filtered using DADA2. Amplicon sequence variants were then aligned with MAFFT, and a phylogenetic tree was generated. Taxonomy was assigned using a Naïve Bayes filtered classifier trained on the Greengenes database, version 13_8, at 99% sequence similarity.