title: Illumina NovaSeq 6000 paired end sequencing; Stool-derived 16s rRNA V4 sequencing from inducible intestinal-epithelium-LSD1-cKO mice (Villin-CreERT2+ Lsd1fl/fl (icKO)) against WT (Villin-CreERT2+ Lsd1fl/+ (icKO)) controls (before and 1-6 weeks after tamoxifen treatment)
design: Stool-derived 16s rRNA V4 sequencing from inducible intestinal-epithelium-LSD1-cKO mice (Villin-CreERT2+ Lsd1fl/fl (icKO)) against WT (Villin-CreERT2+ Lsd1fl/+ (icKO)) controls (before and 1-6 weeks after tamoxifen treatment)
library_name: KO1_6w_p
library_construction_protocol: For stool collection, previously cohabitating mice littermates were separated into individual cages for a duration of 30 minutes. After said period all stool pellets were collected, snap frozen in liquid nitrogen and stored at –80C until further processing. Tamoxifen (Sigma-Aldrich, T5648-1G) was dissolved in 50ml of Corn Oil (Sigma-Aldrich, C8267-500ML) at a 20 mg/ml concentration and stored at 4°C. To induce Lsd1 recombination, 8–12-week-old mice were administered daily via oral gavage with 0.1ml of Tamoxifen for a duration of 5 days. Genomic DNA extraction from frozen stool (snap frozen in liquid N2) samples was done using the QIAamp Fast DNA Stool Mini Kit (Qiagen, Cat. No. 51604) according to protocol. Lysis temperature was adjusted to 95℃. 16S rDNA Amplicon Metagenomic Sequencing of bacterial gDNA derived from mouse stool was outsourced to Novogene UK. Amplicon library of the V4 region was obtained using the 515F (5'-GTGCCAGCMGCCGCGGTAA-3') and 806R (5'- GGACTACHVGGGTWTCTAAT -3') primer pairs connected with barcodes (see sample table for barcodes). The PCR products with proper size were selected by 2% agarose gel electrophoresis. Same amount of PCR products from each sample was pooled, end-repaired, A-tailed and further ligated with Illumina adapters.