title: GSM5700667: Sample 10: EfrCD_etbr_rep1_t0; Enterococcus faecalis; OTHER
library_construction_protocol: Plasmid DNA was extracted by alkaline lysis followed by precipitation in EtOH. The plasmids were purified again using the NucleoSpin Gel and PCR Clean-up Kit (Macherey-Nagel). To extract the efrCD genes, 7 μg plasmid was digested with 40 U of SfiI (NEB), separated on a 1% agarose gel and the band containing the efrCD genes were extracted with the NucleoSpin Gel and PCR Clean-up Kit (Macherey-Nagel). 500 ng DNA was sheared twice in a Covaris microTUBE on a Covaris E220 Focused-ultrasonicator to achieve a distribution with an average fragment length of 150 base pairs using peak power 175, duty factor 10, cycles/burst 200 for 280s at 7°C. In-between cycles the microTUBEs were spun down before second shearing. 150 ng of sheared fragments was used as input for the TruSeq Nano DNA Library Prep Kit (Illumina). Illumina libraries were prepared following the manufacturer's protocol with one modification to yield a narrow fragment distribution optimized for 150 bp inserts by using a ratio of 5.4 parts of sample purification beads to one part of water (135 μl SPB + 25 μl nuclease-free H2O) in the library size selection step.