GSM5746761: HSFA3-ACC+56h-R1; Arabidopsis thaliana; ChIP-Seq
Cross-linking and immunoprecipitation of samples was performed as described previously (Kaufmann et al., 2010; Friedrich et al., 2021). Briefly, 3xFlag-HSFA2 and 3xFlag-HSFA3 seedlings were harvested at the indicated time points (3 replicates) and immediately cross-linked under vacuum in 25 ml ice-cold MC buffer (1 % (v/v) formaldehyde) for 2 x 10 min. For chromatin extraction, frozen tissue was ground, resuspended in 25 ml M1 buffer, and filtered through Miracloth mesh (Merck), washed five times in 5 ml M2 buffer, and washed once in 5 ml M3 buffer with a 10 min, 4°C, 1,000 g centrifugation step in between each washing step. The chromatin pellet was resuspended in 1 ml Sonication buffer and sonified using a Diagenode Bioruptor (17 cycles of 30 sec on/off at low intensity settings). For 3xFlag-HSFA2/A3 ChIP, chromatin was incubated with anti-DYKDDDDK paramagnetic beads (Miltenyi Biotec) for 1.5 h at 4°C and recovered with DYKDDDDK isolation kit (Miltenyi Biotec). DNA libraries for each sample were prepared from isolated chromatin using the NEBNext Ultra II DNA Library Prep Kit for Illumina (NEB) in combination with NEBNext Multiplex Oligos for Illumina (NEB) according to the manufacturer's instructions. No size selection was performed after adaptor ligation. DNA was amplified by PCR with 11 amplification cycles. Library concentration and fragment size distribution were checked using D1000 ScreenTape with TapeStation bioanalyzer (Agilent). Library quantification was done with the NEBNext Library Quant Kit for Illumina (NEB) according to the manufacturer's instructions. Pooled libraries were sequenced on a NextSeq 500 (Illumina), 75 bp SE reads.