Plant sample of Astragalus epiglottis
DNA was extracted from ~20 mg of dry herbarium plant material using the NucleoSpin Plant II kit (Macherey-Nagel, Dren, Germany), following the manufacturers manual with slight modifications. Cell lysis in step 2a was performed using 600 l buffer PL1, no RNase A was added since the plant materials were too old to preserve RNA, and incubation lasted for 1:30h. An extra washing was performed in step 6, adding 350 l buffer PW2 before drying the membrane completely. Finally, DNA was eluted in a 50 l buffer PE (5 mM Tris/HCl, pH 8.5). DNA concentration was measured using an Invitrogen Qubit 4 Fluorometer using the High Sensitivity (HS) assay kit (Thermo Fisher Scientific, Waltham, Massachusetts, USA), and fragmentation was visually evaluated on a 1% agarose gel. Before library preparation, genomic DNA was diluted to 250 ng in 55 l buffer PE and sonicated using a Covaris M220 Focused-ultrasonicator (Covaris, Woburn, Massachusetts, USA) to obtain DNA fragments of about 350 bp. Accurate profiling of the sonicated sample size distribution was performed on an Agilent 4150 TapeStation System using the High Sensitivity D1000 ScreenTape (Agilent Technologies, Santa Clara, California, USA). Libraries were prepared using NEBNext Ultra II DNA Library Prep Kit for Illumina and the NEBNext Multiplex Oligos for Illumina (Dual Index Primers Set 1, New England Biolabs, Ipswich, Massachusetts, USA) and following the manufacturers protocol. In step 3 of the protocol, the size selection of adaptor-ligated DNA was adjusted for each sample according to the average sample size measured with TapeStation. PCR amplification of the adaptor-ligated DNA was performed with eight cycles. Each library was profiled with Qubit and TapeStation assay, as above. Individual libraries were mixed in 16-sample pools, using equal DNA amounts (200 ng or 250 ng) from samples with similar fragment average sizes. Pooled libraries were dried by vacuum centrifugation and resuspended in 7 l nuclease-free water. Target enrichment was performed using the myBaits Hybridization Capture Kits (Daicel Arbor Biosciences, Ann Arbor, Michigan, USA), following the manufacturers protocol v.5.02. A hybridization temperature TH of 60C was chosen. The temperature TW also corresponded to 60C. The enriched libraries were amplified with 10 PCR cycles. After amplification, pooled libraries were purified using NucleoSpin Gel and PCR Clean-up Kit (Macherey-Nagel, Dren, Germany) and characterized by measuring concentration and fragment sizes as described above. The 16-sample pools were again pooled in equimolar quantities and sequenced at the Core Facility Genomics (CF-GEN) of the Helmholtz Zentrum Mnchen, Germany (Deutsches Forschungszentrum fr Gesundheit und Umwelt, GmbH) on an Illumina Nextseq 1000 Sequencing System.