title: Random genomic library prepared from Limulus polyphemus (HSC-2)
design: We extracted DNA from legs or legs+cephalothorax of samples using Qiagen DNeasy kits and adding 2 無 RNase (1 mg/mL) to each extraction. We visualized DNA extracts on 1.5% (w/v) agarose, and we sheared the resulting DNA to 400-500 bp using a Bioruptor (Diagenode, Inc.). 灱fter shearing, we prepared sequencing libraries from 100-500 ng sheared DNA using a commercial library preparation kit (Kapa Biosystems, Inc.) with a set of custom sequence tags to identify each library (Faircloth and Glenn 2012). 阤e amplified 15 無 of each library in a reaction mix of 25 無 Kapa HiFi HS Master Mix (Kapa Biosystems, Inc.), 5 無 Illumina primer mix (5 然 each), 15 無 of adapter-ligated DNA, and 5 無 of ddH2O using a thermal profile of 98 。 for 45 seconds followed by 14 cycles of 98 。 for 15 seconds, 60 。 for 30 seconds, 72 。 for 60 seconds followed by a 72 。 extension for 5 minutes. 灱fter amplification, we cleaned libraries 1:1 with a SPRI-substitute (Rohland and Reich 2012), and we checked the quality of resulting libraries by visualizing 1 無 of each (5 ng/無) on a BioAnalyzer (Agilent, Inc.). 牞ecause we observed adapter-dimer peaks following initial quality control, we cleaned libraries 1-2 additional times using 1:1 SPRI-substitute. 灱fter validating the removal of dimer peaks from the sequencing libraries, we qPCR quantified 5 ng/無 aliquots of each library using a commercial kit (Kapa Biosystems, Inc.), and we combined libraries at equimolar ratios to make a 10 然 pool that we sequenced using PE150 reads on an Illumina HiSeq 2000 (UCSC Genome Technology Center).
identifiers.namespace: SUB1681796
identifiers.id: HSC-2_CTAACGA_L008