Agilent high sensitivity dna analysis kit

Sequencing libraries for DNA methylation mapping were prepared using the µWGBS protocol^{24 (link)}. Starting directly from lysed cells in digestion buffer, proteinase K digestion was performed at 50 °C for 20 minutes. Custom-designed methylated and unmethylated oligonucleotides were added at a concentration of 0.1% to serve as spike-in controls for monitoring bisulfite conversion efficiency. Bisulfite conversion was performed using the EZ DNA Methylation-Direct Kit (Zymo Research, D5020) according to the manufacturer’s protocol, with the modification of eluting the DNA in only 9 µl of elution buffer. Bisulfite-converted DNA was used for single-stranded library preparation using the EpiGnome Methyl-Seq kit (Epicentre, EGMK81312) with the described modifications. Quality control of the final library was performed by measuring DNA concentrations using the Qubit dsDNA HS assay (Life Technologies, Q32851) on Qubit 2.0 Fluorometer (Life Technologies, Q32866) and by determining library fragment sizes with the Agilent High Sensitivity DNA Analysis kit (Agilent, 5067-4626) on Agilent 2100 Bioanalyzer Station (Agilent, G2939AA). All libraries were sequenced by the Biomedical Sequencing Facility at CeMM using the 2x75bp paired-end setup on the Illumina HiSeq 3000/4000 platform.

One μg RNA was used to generate a small RNA sequencing library using reagents and methods provided with TruSeq Small RNA Sample Prep Kit version 2 (Illumina, San Diego, USA). Briefly, T4 RNA ligase was used to ligate RA5 and RA3 RNA oligonucleotides to 5' and 3' ends of RNA, respectively. Adapter-ligated RNA was reverse-transcribed using a RTP primer and the resulting cDNA was amplified in an 11-cycle PCR that used RP1 and indexed RP1 primers. PCR products of 140–160 bp were isolated following electrophoresis through a 6% Novex Tris-borate polyacrylamide gel (Life Technologies). Quality of the generated small RNA sequencing library was confirmed using Agilent High Sensitivity DNA Analysis Kit on Bioanalyzer 2100 instrument. Quadruplexed sequencing of libraries to generate single-end reads of 50 b was performed on Illumina HiSeq 2000 instrument using HiSeq clustering and sequencing reagents (version 2.0). Illumina HCS (version 1.4.8), RTA (version 1.12.4.2), and CASAVA (version 1.8.2) software were used for base-calling and the generation of raw, de-multiplexed sequencing data in FASTQ format. Raw sequencing data was deposited in the NCBI Sequence Read Archive [30 (link)] (accession number SRP047429).

Leaves from SP80-3280 were collected and frozen in liquid nitrogen. Genomic DNA was extracted using DNeasy Plant Mini Kit (Qiagen, Hilden, Germany) following the standard protocol. DNA integrity was analyzed using the Agilent High Sensitivity DNA Analysis Kit (Agilent Technologies, Santa Clara, CA, USA) and Agilent 2100 Bioanalyzer Instrument. Quantification was done using Quant-it^TM PicoGreen® dsDNA Assay Kit (ThermoFisher Scientific, Waltham, MA, USA) and SpectraMax M2 microplate reader (Molecular Devices, San Jose, CA, USA).