fog2_msh2KD_Line45
The PureGene Genomic DNA Tissue Kit (QIAGEN no. 158622) was used for genomic DNA isolation with a supplemental nematode protocol. 2g of each DNA sample was sonicated in 85l TE buffer yielding DNA fragments of 200-400 bp, which were endrepaired (NEBNext end repair module; New England BioLabs) and purified (Agencourt AMPure XP beads; Beckman Coulter Genomics). 3" adenine overhangs were added (AmpliTaq DNA Polymerase Kit; Life Technologies). Custom pre-annealed Illumina adapters were ligated to the fragments. PCR amplification was performed via Kapa Hifi DNA Polymerase (Kapa Biosystems) using Illuminas paired-end genomic DNA primers with 8 bp barcodes. 300-400 bp fractions were selected for excision during size-fractionation of the PCR products on 6% PAGE gels. Gel extraction of the fragments was performed via diffusion at 65C and gel filtration (NanoSep, Pall Life Sciences), followed by a final purification step with Agencourt AMPure beads. The multiplexed DNA libraries were sequenced on Illumina HiSeq 2000 instruments (University of Washington), using default quality filters for the sequence reads. The sequence reads from the 86 genomes were demultiplexed and 100 bp pair-end raw sequence reads were aligned using the Burrows-Wheeler Aligner (BWA) with default settings.