GSM5957867: WT_neg_24; mouse gut metagenome; OTHER
Samples were mixed before centrifugation at 16,100 g for 3 minutes at room temperature. DNA precipitation was performed using 1/10 volume of 3 M NaOAc (pH5.5) and 1 volume Isopropanol to the aqueous phase before overnight incubation at 20o C. DNA precipitate was pelleted at 16,100g for 20 minutes at 4oC before washing in 500 µL of absolute EtOH, centrifugation was performed 16,100 g for 3 minutes at 4oC before DNA pellet was for 15 minutes with no heat (miVac GeneVac on Auto Run setting). Lastly, DNA pellet was resuspended in 100 µL of TE buffer (pH 7) and incubated at 50oC for 30 min. 35 U/ml RNase A was added to the DNA pellet before purification and elution in 40 µL Elution Buffer (QIAquick PCRpurification; QIAGEN). 16S rRNA sequencing of the V4 region and bacterial genome sequencing were performed on an Illumina miSeq using barcoded primers.