FastDigest PstI

Protocol tips

Upstream tips	Protocol tips	Downstream tips
	A total of 250 ng of genomic DNA for each line was double digested with SalI and PstI, PstI and EcoRI, EcoRI and HindIII, or PstI and MspI (FastDigest restriction enzymes; Thermo Fisher Scientific, Waltham, MA, USA); ligated to adapters (Table 1) using the LigaFast Rapid DNA Ligation System (Promega, Madison, WI, USA); and purified using Agencourt AMPure XP (Beckman Coulter, Brea, CA, USA) to eliminate short (<300 bp) DNA fragments.

Protocol tips

A total of 250 ng of genomic DNA for each line was double digested with SalI and PstI, PstI and EcoRI, EcoRI and HindIII, or PstI and MspI (FastDigest restriction enzymes; Thermo Fisher Scientific, Waltham, MA, USA); ligated to adapters (Table 1) using the LigaFast Rapid DNA Ligation System (Promega, Madison, WI, USA); and purified using Agencourt AMPure XP (Beckman Coulter, Brea, CA, USA) to eliminate short (<300 bp) DNA fragments.

Publication protocol

A total of 250 ng of genomic DNA for each line was double digested with SalI and PstI, PstI and EcoRI, EcoRI and HindIII, or PstI and MspI (FastDigest restriction enzymes; Thermo Fisher Scientific, Waltham, MA, USA); ligated to adapters (Table 1) using the LigaFast Rapid DNA Ligation System (Promega, Madison, WI, USA); and purified using Agencourt AMPure XP (Beckman Coulter, Brea, CA, USA) to eliminate short (<300 bp) DNA fragments. Purified DNA was diluted with H2O and amplified by PCR with indexed primers (Table 1 and Supplementary Table S1). The PCR mixture (50 µl) contained 0.4 ng of DNA, 0.2 µM of each indexed primer (one pair per mixture), 1× PCR buffer for KOD –plus– Ver. 2 (Toyobo, Osaka, Japan), 160 µM dNTPs, 1 mM MgSO4, and 1 U DNA polymerase (KOD –plus–; Toyobo). Thermal cycling conditions were as follows: a 3 min initial denaturation at 95°C; 20 cycles of 30 s of denaturation at 94°C, 30 s of annealing at 55°C, and a 60 s extension at 72°C; and a final 3 min extension at 72°C. Amplicons were pooled and separated on a BluePippin 1.5% agarose cassette (Sage Science, Beverly, MA, USA), and fragments of 300–900 bp were purified using the QIAGEN Mini Elute Kit (Qiagen). Concentrations of the resultant libraries were measured using the KAPA Library Quantification Kit (KAPA Biosystems, Wilmington, MA, USA) on an ABI-7900HT real-time PCR system (Life Technologies). Nucleotide sequences of the libraries were determined on a MiSeq (Illumina) in paired-end, 250 bp mode

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Manufacturer protocol

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