Nebnext indexing primers

DNA‐renatured samples were prepared as described above and aliquoted for immunoprecipitation or membrane capture. For membrane capture, approximately 40 μg renatured proteins or fragmented DNA alone (20 μg, negative control) were slowly passed through a 0.2 μm nitrocellulose membrane (1060004, GE Healthcare) pre‐blocked with 250 μg Yeast tRNA (Thermo Fisher Scientific). The membranes were then washed three times with 10 mM Tris–HCl pH 7.5, 100 mM KCl, 0.1% Triton X‐100; twice with 10 mM Tris–HCl pH 7.5, 500 mM KCl, 0.1% Triton X‐100; and once with 1X TE. Captured proteins and nucleic acids were eluted by the addition of 2% SDS/8M Urea and the nucleic acids extracted with Isol (5‐PRIME) as described above. Immunoprecipitation with Aβ 4G8 (1 μg) and mouse IgG (1 μg) were performed as described for RNA immunoprecipitation of renatured proteins above using approximately 40 μg of renatured proteins per IP. Protein A magnetic beads were pre‐blocked for 1 h at RT with 70 μg Yeast tRNA (Thermo Fisher Scientific). Sequencing libraries were prepared using the NEBNext DNA ultra kit (E7370, NEB), using NEBNext Indexing primers (E7335, NEB), and the libraries pooled and sequenced on the MiSeq system (Illumina) using paired‐end reads of 300 bp. Data can be accessed at GEO GSE99127.

Bisulfite sequencing (BS-seq) libraries were constructed using genomic DNA (gDNA) extracted from rosette leaves of 4–5-week-old plants. 500 ng total gDNA was sheared to 100–1000 bp using Bioruptor Pico (Diagenode), then purified with 1.2× volume of SPRI beads (Beckman Coulter, cat. no. A63881). Fragmented gDNA was ligated to NEBNext Adaptor for Illumina using NEBNext Ultra II DNA library prep kit for Illumina (New England Biolabs, cat. no. E7645). We performed bisulfite conversion twice with ligated libraries (QIAGEN, cat. no. 59104) to prevent incomplete conversion (<99% conversion) of unmethylated cytosines. Converted libraries were subjected to SPRI bead purification with 0.8× volume of beads. We amplified bisulfite-converted libraries with NEB next indexing primers (New England Biolabs Inc, cat. no. E7335S).