Nebnext ultra dna library prep for kit

Genomic DNA was quantified using Qubit® dsDNA BR (Invitrogen, Life Technologies) and its integrity was assayed with an agarose gel. gDNA fragmentation was done using the Covaris S2 instrument (Covaris Inc.) adjusting the settings as follows: 10% duty cycle, intensity 5, and 200 cycles per burst for 40 s (for 600 bp size distribution). Libraries were prepared using the NEBNext® Ultra DNA Library Prep for Illumina® kit (ref. E7370) according to the manufacturer's protocol. Briefly, 100 nanograms of DNA were subjected to end repair, addition of ‘A’ bases to 3’ ends, ligation of adapters and USER excision. All purification steps were performed using AgenCourt AMPure XP beads (ref. A63882, Beckman Coulter). Library amplification was performed by PCR using NEBNext® Multiplex Oligos for Illumina (Index Primers Set 1, ref. E7335), (Index Primers Set 2, ref. E7500), (Index Primers Set 3, ref. E7710) or/and (Index Primers Set 4, ref. E7730). Final libraries were analyzed using Agilent DNA 1000 chip to estimate the quantity and check size distribution, and were then quantified by qPCR using the KAPA Library Quantification Kit (ref. KK4835, KapaBiosystems) prior to amplification with Illumina's cBot. Libraries were sequenced 2 × 151 + 8 bp on Illumina's NextSeq500. Whole genome sequencing data are available at Array Express under accession E-MTAB-11600.