Nextera dna sample preparation kit

Chromatin collection and chromatin immunoprecipitation were performed as described previously (Nègre et al. 2011 (link)). Transgenic lines containing GFP-tagged transcription factors within their endogenous genomic contexts were produced using the P[acman] bacterial artificial chromosome (BAC) system as previously described (Venken et al. 2009 (link); Roy et al. 2010 (link)). Antibody details are available at modMine (http://intermine.modencode.org). A number of antibodies were generous contributions from members of the Drosophila research community: Ken Cadigan (Pan/TCF), Andy Dingwall (Cmi/Lpt), Eric Lai (Insv), Erika Bach (Stat92E), Jim Kadonaga (TBP), Ken Irvine (Yki), Claude Desplan (Prd), Stephen Crews (Sc), Sean Carroll (Dll), Richard Mann (Hth), and Scott Hawley (Trem). Immunoprecipitated DNA was prepared for Illumina sequencing either as described in Nègre et al. (2011) (link) or using the Epicentre Nextera DNA Sample Preparation Kit. Briefly, Nextera library preparations were performed using the High Molecular Weight tagmentation buffer, and tagmented DNA was amplified using 12 cycles of PCR. DNA was then sequenced on an Illumina HiSeq 2000 according to the manufacturer’s standard protocols.

The genomic DNA was processed using the Nextera DNA sample preparation kit (Epicentre Biotechnologies, Madison, WI). Thirty nanograms of sample DNA were fragmented utilizing 5 μl of Tagment DNA Enzyme with 25 μl of Tagment Buffer. Tagmentation reactions included in the Nextera kit were performed by incubating the sample for 5 min at 55°C. The tagmented DNA was purified using the QiagenMinElute protocol (QIAGEN, Germany). Purified DNA was eluted from the column with 11 μl of nuclease-free water. Purified DNA (5 μl) was used as the template in a 20-μl volume for limited-cycle PCR (5 cycles) and processed as outlined in the Nextera protocol (Illumina). Amplified DNA was purified using the AMPure Bead cleanup (Beckman Coulter, USA) according to the manufacturer's protocol. The fragment size distribution of the tagmented DNA was analyzed utilizing a 2100 Bioanalyzer with a 7500 DNA assay kit (Agilent Technologies, Santa Clara, CA). Fragments of ~600 bp long were carried out for sequencing. The library was sequenced on one lane of an Illumina HiSeq2000 instrument to generate paired-end reads. Sequencing was performed by The Vincent J. Coates Genomics Sequencing Laboratory (GSL) at the University of California, Berkeley.

Illumina sequencing libraries were constructed from P5 and P5-EM. Genomic DNA was extracted with the Hoffman-Winston DNA preparation method. Bar-coded DNA fragment libraries were prepared by a Nextera DNA sample preparation kit (Epicentre Biotechnologies, Madison, WI) following standard procedures and published recommendations [59 ].
Briefly, 50 ng of yeast genomic DNA from each strain were tagmented (tagged and fragmented) by the Nextera transposome. The tagmented DNA was purified following the AMPure (Agencourt) purification protocol. Purified tagmented DNA libraries were PCR-amplified with the Nextera PCR Master Mix. PCR-amplified libraries were cleaned following the AMPure (Agencourt) purification procedures, and submitted for sequencing.

Genomic DNA of VP152 strain was extracted using Masterpure^TM DNA purification kit (Epicenter, Illumina Inc, Madison, WI, USA) and subjected to RNase (Qiagen, USA) treatment (Ser et al., 2015 (link)). The DNA quality was quantified using NanoDrop spectrophotometer (Thermo Scientific, Waltham, MA, USA), and a Qubit version 2.0 fluorometer (Life Technologies, Carlsbad, CA, USA). Illumina sequencing library of genomic DNA was prepared using Nextera^TM DNA Sample Preparation kit (Illumina, San Diego, CA, USA) and library quality was validated by a Bioanalyzer 2100 high sensitivity DNA kit (Agilent Technologies, Palo Alto, CA, USA) prior to sequencing. The genome of VP152 strain was sequenced on MiSeq platform with MiSeq Reagent Kit 2 (2 × 250 bp; Illumina Inc, San Diego, CA, USA). The trimmed sequences were de novo assembled with CLC Genomic Workbench version 5.1 (CLC Bio, Denmark).

Firstly, soil samples were homogenized by sieving (2 mm) to remove rocks and plant material. Total community DNA was directly extracted using PowerMax Soil DNA Isolation Kit (Mo Bio Laboratories, United States), following manufacturer’s instructions. DNA integrity was checked on 1% agarose gel electrophoresis. DNA quality was measured using NanoDrop spectrophotometer (Thermo Scientific, United States) at A260/280 nm ratio. DNA samples were used to prepare libraries with the Nextera^TM DNA Sample Preparation Kit (Illumina^®-compatible). Environmental DNA samples from each site were barcoded for shotgun sequencing in one single lane of Illumina HiSeq 2000 platform (2 × 100 bp) at Multi-User Laboratory of Functional Genomics (Piracicaba, Brazil), according to the manufacturer’s instructions.

Chloroplasts were isolated from A. hirsuta and A. nipponica as described in Okegawa and Motohashi [43 (link)]. DNA was isolated from the chloroplasts using the DNeasy Plant Mini Kit (Qiagen, Valencia, CA, USA), while the total DNA was isolated from leaves of A. flagellosa. NGS libraries were constructed using the Nextera DNA Sample Preparation Kit (Illumina, San Diego, CA, USA) and sequenced as single-ended reads using the NextSeq500 platform (Illumina). About 2 Gb (1.4 Gb, 12 M clean reads) of sequences were obtained for A. flagellosa (43 Mb mapped reads, 282.69× coverage). Additionally, 400 Mb (300 Mb, 2.5 M clean reads) were obtained for both A. hirsuta (64 Mb mapped reads, 417.17× coverage) and A. nipponica (72 Mb mapped reads, 455.87× coverage). The generated reads were assembled using velvet 1.2.10 [44 (link)] and assembled into complete chloroplast genomes by mapping to previously published whole chloroplast genome sequences. Sequence gaps were resolved using Sanger sequencing. Genes were annotated using DOGMA [45 (link)] and BLAST. The newly constructed chloroplast genomes were deposited in the DDBJ database under the accession numbers LC361349-51. Finally, the circular chloroplast genome maps were drawn using OGDRAW [46 (link)].