16S_V4_amplicon_of_poultry_litter_metagenome_Trt1_replicate_4
Library preparation and sequencing were conducted at the University of Missouri Genomics Technology Core. Prokaryotic 16S rRNA amplicons were generated by targeting the V4 region of the 16S rRNA gene with universal primers U515F/806R flanked by Illumina adapter sequences (Caporaso et al., 2011; Walters and Caporaso et al., 2011). PCRs (50 uL) contained 100 ng template DNA, 0.2 uM of each primer, 200 uM each dNTP, 1 U Phusion High-Fidelity DNA Polymerase (Thermo Fisher Scientific, Cat #F530S), and 1X Phusion HF Buffer (Thermo Fisher Scientific, Cat #F518L). Cycling conditions were 98 C for 3 min; 25 cycles of 98 C for 15 s, 50 C for 30 s, and 72 C for 30 s; with a final extension at 72 C for 7 min. Amplicons (5 uL per reaction) were pooled, mixed thoroughly, and purified with Axygen Axyprep MagPCR Clean-up Beads (Thermo Fisher Scientific, Cat #14-223-227) at a 1:1 bead:amplicon ratio, incubated 15 min at room temperature, washed twice with 80% ethanol, air-dried, and resuspended in 32.5 uL EB buffer (Qiagen, Cat #19086); the tube was placed on a magnetic stand for 5 min to recover the supernatant. The pooled library was assessed on an Advanced Analytical Fragment Analyzer (Agilent Technologies), quantified with the Quant-iT High Sensitivity dsDNA Assay Kit (Thermo Fisher Scientific, Cat #Q33120), and sequenced on an Illumina MiSeq as 2x250 bp paired-end reads with dual indexing.