Er81050

DNase-seq assays were performed as described^{30 (link)}. Briefly, 10,000 cells from each cell type were collected by FACS sorting. 0.3 unit of DNase I (Roche) was added to each cell type and incubated at 37 °C for 5 min. Reactions were stopped by adding 80 μl of stop buffer (10 mM Tris-HCl, 10 mM NaCl, 0.1% SDS, 10 mM EDTA) containing 1 μl of 20 mg/ml proteinase K. Samples were incubated at 65 °C overnight, and DNAs were purified by using MinElute reaction cleanup kit following the manufacturer’s protocol. Purified DNA was end-repaired using an end-repair kit (ER81050, Epicenter), followed by A-addition using Klenow fragment (3’ to 5’ exo minus) and dATP. The Y-shaped adapters were then ligated to DNA ends using T4 DNA ligase. Libraries were amplified with Illumina primers for 16 cycles and library fragments from 150 bp to 300 bp were isolated for sequencing on Illumina HiSeq or NovaSeq platforms.

For each accession, three plants were pooled and total RNA was extracted by TRIzol (Invitrogen, Carlsbad, California, 15596-018), DNase treated and mRNA purified with oligo dT Dynabeads (Life Technologies, Carlsbad, California). RNA was then fragmented using Ambion Fragmentation buffer (Life Technologies) and first and second strand cDNA synthesis was carried out using Invitrogen kit 18064-071. The ends of sheared fragments were repaired using Epicentre (Madison, Wisconsion) kit ER81050. After A-tailing using exo-Klenow fragment (New England Biolabs, Ipswich, Massachusetts, NEB M0212L), barcoded adaptors were ligated with Epicentre Fast-Link DNA Ligation Kit (Epicentre LK6201H). Adaptor-ligated DNA was resolved on 1.5% low melt agarose gels for 1 hr at 100 V. DNA in the range of 200–250 bp was excised from the gel and purified with the Zymoclean Gel DNA recovery kit (Zymo Research). The libraries were amplified by PCR for 15 cycles with Illumina PCR primers 1.1 and 1.2 with Phusion polymerase (NEB F-530L).
Single-end 32 bp sequencing was performed at the University of Southern California Epigenome Center on an Illumina (San Diego, California) GAIIx instrument using fourfold multiplexing.