male  PCB, replicate 6
Microbial genomic DNA was isolated from mouse fecal samples. Libraries were prepared following Illuminas 16S library preparation guide and Illuminas Nextera Index Kit (FC-121-1012). Samples were diluted to 5 ng/L in 10mM Tris-HCl pH 8.5. The 16S variable region was amplified using 12.5 ng (in a volume of 2.5 L) microbial genomic DNA. PCR conditions:  95C for 3 minutes; 25 cycles of 95C for 30 seconds, 55C for 30 seconds, and then 72C for 30 seconds; 72C for 5 minutes. 16S Amplicon PCR Forward Primer = 5'TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGCCTACGGGNGGCWGCAG 16S Amplicon PCR Reverse Primer = 5' GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGACTACHVGGGTATCTAATCC. Amplicon PCR CleanUp using AMPure XP beads. Samples stored in  20 C overnight. Dual indices and Illumina sequencing adapters were attached using the Nextera XT Kits A and D (FC-121-1012). 5. Index PCR Clean-Up  was performed using SpriSelect beads.  2 l of the sequencing libraries was aliquoted and mixed to make the pooled library. DNA concentration in ng/l obtained from the Qubit assay was calculated into nM using the following formula: (concentration in ng/l) / (660 g/mol  average library size)  1,000,000 = concentration in nM. The pooled library was diluted to 10 nM using Tris-HCl 10 mM, pH 8.5. The 10 nM pooled library was diluted to 4 nM Tris-HCl 10 mM, pH 8.5 with 0.1% Tween 20. The 4 nM pooled library was denatured with 0.2N NaOH for 5 min at RT, and then diluted with the hybridization buffer to 20 pM. The diluted 20 pM pooled library was further diluted to 10 pM using the prechilled hybridization buffer. PhiX control library was denatured with 0.2N NaOH, and then diluted with the hybridization buffer to 20 pM. The diluted 20 pM PhiX control library was further diluted to 10 pM using the prechilled hybridization buffer. 420 ul of the final diluted 10 pM pooled library was combined with 180 ul of the final diluted 10 pM PhiX control library.  The combined sample library and PhiX control was heat denatured at 96C for 2 minutes, then cooled in an ice water bath for 5 minutes before MiSeq sequencing.  Sequencing was done on a Nano-300 cycle test chip (MS-103-1001) to confirm sample concentration followed by Illumina MiSeq Reagents kit v3 (600 cycles) (MS-102-3003) at 10 pM and 30% PhIX.
SUB14767360
MC_BW_16_M_PCB_16