title: GSM5225350: 2k_1_M9_biofilm; Enterococcus faecalis OG1RF; OTHER
library_construction_protocol: Substrates were removed from the CDC biofilm reactor chamber and processed to remove adherent biofilm cells.  Biofilms from multiple substrates from each reactor were pooled in a conical tube, pelleted at 6371× g for 15 min, and frozen at -80 oC until further use.  Pellets were resuspended in 180 uL enzymatic lysis buffer (20 mM Tris-HCl pH 8.0, 2 mM EDTA, 1.2% Triton X-100) with 30 mg/mL lysozyme and 500 U/mL mutanolysin.  After 30 min incubation at 37 oC, 25 uL Proteinase K and 200 uL Buffer AL (DNeasy Blood and Tissue Kit, Qiagen) were added.  Tubes were incubated at 55 oC for 30 min, after which DNA was extracted using a DNeasy Blood and Tissue Kit following manufacturer's instructions. DNA was sheared and processed using the Illumina TruSeq Nano library preparation kit.  The libraries were normalized to 2 ng/μl, and 10 ng of each library was used as a PCR template to enrich for the mariner transposon junctions using a transposon-specific primer (mariner-seq) and the Illumina P7 primer.  1 μl of the enrichment PCR product was diluted 100-fold, and 10 μl of the diluted DNA was used as template for the indexing PCR (TruSeq P5 indexing primer + P7 primer).  The final libraries had unique combinations of P5 and P7 indexes suitable for multiplexed sequencing.  Libraries were quantified, normalized, and pooled.  The pooled library was cleaned using 1×SPRI beads (Ampure XP, Beckman Coulter) and eluted in 1:10 TE buffer (made from 1×TE buffer purchased from Fisher).  The amplicon size distribution of the pool was verified on a Bioanalyzer (Agilent) and the concentration of functional library molecules calculated using Kapa Library Quantification kit for Illumina.  The pool was sequenced using an Illumina HiSeq 2500 in 125-bp paired end output mode at the University of Minnesota Genomics Center.