title: NextSeq 500 sequencing; The effect of γ-butyrolactone (MB3) treatment on the chromatin landscape after wounding of Arabidopsis thaliana roots
design: The effect of γ-butyrolactone (MB3) treatment on the chromatin landscape after wounding of Arabidopsis thaliana roots
library_construction_protocol: Samples were taken from roots of 7 days old plants in triplicates. To apply wound stress, roots were cut into 3 mm explants and harvested at 0, 3 and 6 h after wounding. Plants were aligned on Murashige-Skoog (MS) media containing 1% sucrose and 0.6% (w/v) gelzan, overlaid with a nylon mesh (Sefar) and grown for 7 days under continuous light conditions at 22°C. All samples were treated with gamma-butyrolactone (MB3, CAS No. 778649-18-6, ab141255, Abcam) (CHEBI: 42639) 100 micromolar was applied 24 h before cutting by transferring the nylon mesh to MB3-containing MS plates. Root samples were ground to a fine powder using a multi-beads shocker (MB1200, Yasui Kikai) and the nuclear fraction was isolated after cross linking with 1% formaldehyde for 10 min. Chromatin was sheared at 4–6°C for 15 min with a focused ultrasonicator (Covaris) with the following settings: duty cycle 5%, intensity 4, and cycles per burst 200. Chromatin was immunoprecipitated using antibodies against H3 (ab1791; Abcam), H3K27me3 (07-449; Millipore), H3K4me3 (ab8580; Abcam), H3K9/14ac (06-599, Millipore) and H3K27ac (ab4729, Abcam).  DNA was extracted with phenol-chlorophorm (1:1), precipitated with ethanol and dissolved in TE.   Libraries were prepared using the KAPA Hyper Prep Kit for Illumina (KK8502, KAPA Biosystems) and Illumina compatible adaptors (E7335, E7500, E7710, E7730, NEB).