Biomek fxp

Anchored Hybrid Enrichment data were collected through the Center for Anchored Phylogenomics at Florida State University (www.anchoredphylogeny.com) following the general methods of Lemmon et al. [30 (link)] and Prum et al. [59 ]. Briefly, each genomic DNA sample was sonicated to a fragment size of ~175-325 bp using a Covaris E220 Focused-ultrasonicator with Covaris microTUBES. Subsequently, library preparation and indexing were performed on a Beckman-Coulter Biomek FXp liquid-handling robot following a protocol modified from Meyer and Kircher [60 (link)]. One important modification is a size-selection step after blunt-end repair using SPRIselect beads (Beckman-Coulter Inc.; 0.9x ratio of bead to sample volume). Indexed samples were then pooled at equal quantities (approximately 16 samples per pool), and enrichments were performed on each multi-sample pool using an Agilent Custom SureSelect kit (Agilent Technologies), described herein as the Spider Probe Kit, that contained the probes designed for AHE loci from the spider genomic data detailed above. After enrichment, each set of reactions were pooled in equal quantities for sequencing on three PE150 Illumina HiSeq2500 lanes. Sequencing was performed in the Translational Science Laboratory in the College of Medicine at Florida State University.

Bacterial liquid cultures were mixed using a Biomek FXp liquid handling station (Beckman Coulter) into group-based matrix plates and position-based matrix plates as indicated in Fig. 3. The first PCR amplifies the sgRNA sequence and adds one of 12 possible barcodes specific for the individual pool plates using the primers gRNA_fwd_1-gRNA_fwd_12 and gRNA_rev (barcode 1 in Fig. 3). The second PCR amplifies the first PCR, while adding 96 individual barcode combinations and an Illumina specific sequencing linker (barcode 2 and 3 in Fig. 3). For all primer sequences see Supplementary Table S6. Deconvolution was performed using a custom-written javascript program.