Dna clean concentrator 5 column

Whole-genome amplification was performed on single flow-sorted nuclei using 10 units of Φ29 polymerase and 10× Φ29 buffer (NEB cat#M0269L), 1 mM dNTP (GE Healthcare, cat#28-4065-51), and 50 μM random hexamer (phosphorothioate modification on the two 3’-terminal nucleotide - NNNN*N*N - synthesized by Sigma Aldrich) to each well. The total 50 μL reactions were mixed by gently pipetting up and down and spinning down the reaction. Reactions were incubated at 30°C for 120 min to obtain approximately 500 ng of DNA. The polymerase was denatured subsequently at 65°C for 3 min. The amplified DNA was purified using DNA Clean & Concentrator-5 columns (Zymo D4004).

TC, LD, and WZS libraries were constructed by mixing equal amounts DNA from three individuals within each breed following a previously described protocol. Genomic DNA was sonicated to generate fragments of approximately 100–500 bp using a Covarias sonication system. Libraries were constructed using a Paired-End DNA Sample Prep kit (Illumina, CA, USA) according to the manufacturer’s instructions. Adaptor-ligated DNA was used for subsequent MeDIP enrichment with an antibody that specifically recognizes 5′-methylcytosine. The enriched methylated fragments were purified on DNA Clean & Concentrator-5 columns (Zymo Research, Orange, CA, USA). DNA from qualifying MeDIP experiments was amplified by adaptor-mediated PCR to produce libraries with insert sizes between 220 and 300 bp. Amplification quality and quantity were evaluated using an Agilent 2100 Analyzer and the DNA 1000 Nano Chip Kit (Agilent Technologies, Santa Clara, CA, USA). Each MeDIP library was subjected to paired-end sequencing with 49-bp reads on an Illumina Hiseq 2000 Sequencing System (Illumina, San Diego, CA, USA) following the manufacturer’s instructions (BGI, Shenzhen, China).

Genomic DNA was extracted from drought-stressed and control DK151 and IR64 plants using the AxyPrep Multisource Genomic DNA Miniprep Kit (Axygen Biosciences). Each sample consisted of three biological replicates. Libraries prepared using non-immunoprecipitated “input” DNA from three biological replicates were sequenced as controls. The MeDIP-seq DNA libraries were prepared according to the protocol described by Li et al. (2010) (link). Briefly, 5 μg DNA from each sample was sonicated to produce DNA fragments (100–500 bp). Libraries were then constructed using the Paired-End DNA Sample Prep Kit (Illumina, San Diego, CA, USA). Adapter-ligated DNA was immunoprecipitated using a monoclonal anti-methylcytidine antibody. The specificity of the enrichment was confirmed by quantitative reverse transcription polymerase chain reaction (PCR) using the SYBR Green Master Mix (Applied Biosystems). The enriched methylated fragments and input DNA were purified with DNA Clean & Concentrator-5 columns (Zymo). The purified DNA was then used as the template for adapter-mediated PCR. Amplicon quality and quantity were evaluated using the 2100 Analyzer DNA 1000 chips (Agilent). Ultra-high-throughput 50PE sequencing was conducted using the Illumina HiSeq 2000 system. Raw sequencing data were processed by the Illumina base-calling pipeline.

For each developmental stage, the tooth germs were isolated separately from three randomly selected embryos with a specific sex (male) as biological replicates. MeDIP DNA libraries were constructed following the protocol as described previously [47 (link)]. In brief, genomic DNA was extracted using the DNeasy Blood & Tissue Kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. Five microgram DNA was sonicated to fragments ranging from 100 to 500 bp. Subsequently, DNA underwent end-repair, the generation of 3’-dA overhangs, and adaptor ligation steps using Paired-End DNA Sample Prep kit (Illumina, San Diego, CA, USA). Adaptor-ligated DNA was then immunoprecipitated by anti-5-methylcytosine monoclonal antibody (Diagenode, NJ, USA). The enriched methylated fragments and 10 % input DNA were purified on DNA Clean & Concentrator-5 columns (Zymo, CA, USA) according to the manufacturer’s manuals. Enriched fragments were amplified by adaptor-mediated PCR, the products were quantified on Agilent 2100 Analyzer (Agilent Technologies, Santa Clara, CA, USA), and MeDIP library was sequenced on an Illumina HiSeq 2000 Sequencing System by Beijing Genomics Institute (BGI, Shenzhen, China). After the completion of sequencing run, raw data was processed by the Illumina base-calling pipeline.

The Golden variety of A. hortensis was grown hydroponically in a growth chamber at BYU as previously described. Plants were dark-treated for 72 h at which point young leaf tissue was harvested and extracted for high molecular weight (HMW) genomic DNA using the Qiagen (Germantown, MD) Genomic-tip protocol. The DNA concentration was checked using the dsDNA High Sensitivity DNA Assay on the Qubit^® 2.0 Fluorimeter (Invitrogen, Merelbeke, Belgium).
Samples for DNA sequencing were prepared with and without fragmentation using Covaris g-TUBEs (Woburn, MA) and the ZYMO DNA Clean & Concentrator-5 column (Irvine, CA, United States). Samples were fragmented using both the ZYMO DNA kit and Covaris g-TUBEs following manufacturer’s instructions. Samples prepared with the Covaris g-TUBEs were fragmented at several centrifugation speeds, including 3,800, 4,000, and 4,200 RPM. In total, nine libraries from the original DNA stock were prepared for sequencing using the 1D Genomic DNA by Ligation MinION library preparation kit. Libraries were sequenced on R9 flow cells on a MinION for 48 h using MinKNOW 2.0 software with the following settings: DNA, PCR-free, no multiplexing, SQK-LSK109 kit (Oxford Nanopore Technologies, Ltd., Oxford, United Kingdom). No alterations were made to voltage or time. Albacore v2.3.1, part of the MinKNOW package, was used for base calling.