Auto hmedip kit

Oxidative bisulfite treatment (oxBS) of 1 μg of DNA was performed with TrueMethyl oxBS Module (Nugen), as described in (29 (link)). Levels of 5-hydroxymethylcytosine (5hmC) were measured by pyrosequencing and they were calculated by subtracting the proportion of Cs obtained in oxBS from BS (5hmC% = 5mC%_BS – 5mC%_oxBS). For hydroxymethylated DNA immunoprecipitation (hMeDIP) total DNA was extracted using the Gentra Puregene kit (Qiagen), 1.25 μg of which were sonicated for 6 cycles [15 s ON/90 s OFF] on a Bioruptor Pico. 5hmC was immunoprecipitated using 2.5 μg of anti-5hmC antibody as per the manufacturer's instructions (Auto hMeDIP Kit, Diagenode). The enrichment was measured using qPCR that was performed as previously described in (28 ) following these conditions: denaturation 95°C 5min, amplification [95°C 15 s, 60°C 60 s, 72°C 60 s] × 40 cycles, melting curve 95°C 60 s, 55–90°C with 0.5°C increment every 5 s. The percentage of precipitated 5hmC over input was calculated as such as the result of 2^[(Ct(_10%input) – 3.32) – Ct(_hmetDNA-IP)] × 100%.

Genomic DNA was isolated from WT and j-2 (LA3899) mutant flower pedicels as previously described and fragmentation was performed using Diagenode Bioruptor 200 UCD-300 (30 s then off 90 s for 25 cycles, low power position). Following steps were performed using Diagenode Auto hMeDIP KIT in the SX-8G IP-Star® Compact System. Anti-5-methylcytosine antibody (NA8133D3, Merck Millipore, Diagenode) was used for precipitation. DNA was then purified using Auto Ipure kit v2 (Diagenode). MeDIP-qPCR was performed by the same methods as the RT-qPCR using the purified immunoprecipitated DNAs as templates. Primers used for MeDIP-qPCR are listed in Supplementary Table 2.

Genomic DNA was isolated from roots and leaves of the cultivar Charentais (Cucumis melo L. subsp. melo var cantalupensis) using E.Z.N.A Plant DNA Kit (Omega). Fragmentation was performed using Diagenode Bioruptor 200 UCD-300 (30 s then off 90 s for 25 cycles, low power position). The following steps were performed using Diagenode Auto hMeDIP Kit in the SX-8G IP-Star® Compact System. Anti-5-methylcytosine antibody (NA8133D3, Merck Millipore, Diagenode) was used for precipitation. DNA was then purified using Auto Ipure kit v2 (Diagenode). Libraries were synthetized using NebNext Ultra DNA Library Preparation Kit (NEB) according to the manufacturer’s instructions and sequenced by Illumina technology. The same melon accession was used to collect the the total RNA from roots of 10 day old (cultivated in vitro) and from leaves of 3 weeks old, grown in a growth chamber (long day conditions, temperature: 27 °C (day) and 21 °C (night), relative humidity: 60%). The Nucleospin RNA kit (Macherey-Nagel) was used for the extraction according to the manufacturer’s instructions. Libraries were synthetized from 2 µg of total RNA using NEBNext Ultra Directional RNA library Preparation Kit (NEB) according to the manufacturer’s instructions. Two biological replicates were analysed for each tissue. Libraries were sequenced by Illumina technology.