title: GSM8485779: Whole rosette tissue, wild type, 21 day, H2A.Z, ChIP, rep2; Arabidopsis thaliana; ChIP-Seq
library_construction_protocol: 4 g of 21-day-old whole rosette tissue were collected for each biological replicate. Nuclear lysis buffer was replaced with 1.7 mL of ChIP Lysis Buffer (50 mM HEPES-NaOH (pH =7.4), 16.7 mM Tris-HCl (pH = 8), 1 mM EDTA (pH = 8), 150 mM NaCl, 0.1% SDS, 1% Triton X-100, 0.1% Sodium deoxycholate, 1 cOmpleteTM mini tablet / 10 mL solution). The total chromatin samples were sonicated using Bioruptor Pico. Sheared chromatin samples were stored at -80 °C overnight prior to the immunoprecipitation. The following antibodies were used in the analysis: Anti-H3 (ab1791, Abcam) (1:200 dilution), Anti-H3K27me3 (07-449, Millipore) (1:100 dilution), and Anti-H2A.Z (1.5 µg for a 500 µL IP sample) (Deal et al., 2007). The QIAquick MinElute PCR purification kit was replaced with ChIP DNA Clean & Concentrator purification kit (D5205, Zymo research) for elution of the ChIP DNA. The eluted ChIP DNA was quantified us a Qubit 2.0 Fluorometer and used immediately in library construction. 0.5 ng eluted ChIP DNA was used for each library construction. ChIP DNA libraries were constructed using a NEBNext Ultra II DNA Library Prep Kit (NEB; catalog no. E7645L) according to the manufacturer's manual. Illumina Multiplexing adapter was used for indexing. Pair-ended,150-bp sequencing was performed using an Illumina Novaseq S4 lane.