Nextera mp kit

Long insert mate pair libraries of 11 LGL patients (S1-S11) were prepared and sequenced at the Duke Center for Genomic and Computational Biology using the Illumina Nextera MP kit. Sequencing was performed on a HiSeq and read length was 125 bp. Paired-end sequencing of 48 LGL patients (including patients S1-S8) was conducted by Illumina. Patients S9, S10, and S11 were paired-end sequenced at Penn State by Dr. Stephan Schuster.

For mate pair library construction, nuclear DNA from CS + 7EL seedlings was purified as described in [63 (link)] with the addition of a centrifugation step (2 min, 55 rcf) to remove cellular debris after the first resuspension of nuclei. All mate pair libraries aside from the 40 kb library were prepared with the Nextera MP kit (Illumina, San Diego, CA) using the gel-based protocol with the following modifications. Two pairs of tagmentation reactions with 16 μL of tagment enzyme and 6 or 8 μg of nuclear DNA were incubated in 400 μL reactions. Pairs of reactions were pooled and separated by field inversion gel electrophoresis (0.6% Megabase gel, 14.5 h). The size-resolved DNA was divided into 12 fractions and purified using a Zymoclean large fragment DNA recover kit (Zymo Research, Irvine, CA) according to manufacturer’s instructions. Five of the fractions gave a yield greater than 600 ng and were divided for subsequent steps to make distinct libraries. Libraries were amplified with 10, 12 or 15 cycles on the basis of the insert size and amount of DNA used for circularization. The 40 kb mate pair library was constructed using the NxSeq 40 kb Mate Pair Cloning Kit (Lucigen, Middleton, WI) according to manufacturer’s instructions.