Microbiota changes in a model of unpredictable maternal separation combined with unpredictable maternal stress (MSUS)

2 groups of mice, Control and a model of unpredictable maternal stress and maternal separation (MSUS). Stress treatment occurs between day 1 and 15 after birth. Feces from each mice are taken at post natal day 21-24 (T2) and at three months (T3). Genomic DNA was extracted from approximately 20-40 mg of feces from timepoint 2 and timepoint 3 using a QIAamp PowerFecal DNA Kit (Qiagen, Hilden, Germany). After extraction, DNA was eluted in 50 ul of RNase-free water and quality was measured by NanoDrop and Qubit to check the requirements for subsequent sequencing. Library preparation was performed using the Kinnex 16S rRNA kit for full-length 16S sequencing, and sequenced on a PacBio Sequel II instrument. The 27F:AGRGTTYGATYMTGGCTCAG and 1492R:RGYTACCTTGTTACGACTT universal primer set was used to amplify the full-length 16S rRNA gene from the genomic DNA. Both, the forward and reverse 16S primers were tailed with sample-specific PacBio barcode sequences to allow for multiplexed sequencing.