Ovation ultralow v2 kit

Cell growth and crosslinking were done in the same fashion as in ChIP-seq experiments. Generally, we followed the protocol in Rodriguez et al., 2014 (link), with changes described here. Cells were spheroplasted using 10 mg zymolyase (100T, AMSBIO, 120493–1) per 100 OD₆₆₀ cells. For Q cells, zymolyase treatment could take up to 2 hr. We monitored the cells via microscopy and stopped the spheroplasting step when ~80% of the cells were spheroplasted. MNase digestion was performed as described in Rodriguez et al., 2014 (link). High digests (80% mononucleosomes) required 50 U of MNase (Worthington, LS004798), and for the low digests, chromatin was treated with 10 U of MNase. From this step, chromatin was reverse crosslinked as described in Rodriguez et al., 2014 (link). Following reverse crosslinking, RNase, and proteinase-K digestion, DNA was phenochloroform extracted. Any large, uncut genomic DNA species was separated out using Ampure beads (Beckman). Sequencing libraries were generated from the purified DNA using the Ovation Ultralow v2 kit (NuGEN, 0344). Libraries were subjected to 50 bp paired-end sequencing on an Illumina HiSeq 2500 at the Fred Hutchinson Cancer Research Center genomics facility.

Bisulfite PCR sequencing was performed as previously described (10 (link), 23 (link)). Genomic DNA was extracted using the CTAB-based method followed by sodium bisulfite treatment of the DNA by the Qiagen EpiTect bisulfite kit per manufacturer’s instructions. Libraries were generated from purified PCR products amplified from the bisulfite-treated DNA by using previously described primers (10 (link)), using an Ovation Ultralow V2 kit (NuGEN) or a Kapa Kit (Roche) in combination with TrueSeq LT barcodes or IDT UD barcodes (Illumina). Illumina HiSeq 2500 was used to sequence the libraries. Bismark was used to map and process the bisulfite PCR reads (24 (link)). As with the WGBS analysis, reads with three or more consecutive methylated CHH sites were discarded since they are likely to be unconverted reads.

BS-PCR-seq was performed as previously described^{34 (link)}. Briefly, leaf tissues from adult plants of representative T2 lines containing the ZF-SssI transgene were collected and DNA was extracted following a CTAB-based method. Bisulfite conversion was done using the Epitect Bisulfite Conversion kit (QIAGEN). The following regions of FWA were analyzed: Region 1 (chr4: 13038143-13038272), Region 2 (chr4: 13038356-13038499), and Region3 (chr4: 13038568-13038695); which cover fragments of the promoter and 5′ transcribed region of FWA. Pfu Turbo Cx (Agilent) was used to amplify bisulfite-treated DNA using primers containing Illumina adaptors. The primers used are listed in Supplementary Data 10.
For each sample, individual PCR products from each of the three FWA regions were pooled and purified using AMPure beads (Beckman Coulter) before making the libraries. Libraries were made from purified PCR products using a TruSeq Nano DNA Library Prep kit for Neoprep automated library preparation machine (Illumina), a Kapa DNA hyper kit (Kapa Biosystems) with Illumina TruSeq DNA adapters, or an Ovation Ultralow V2 kit (NuGEN).