Amm99021

5mC antibodies (mouse clone 33D3, AMM99021, Aviva Systems Biology) used in MeDIP bind to 5mC in a single stranded DNA, thus, before immuno-precipitation, DNA from cells was sheared to small fragments (∼300 bp) by ultrasound and melted by boiling. 1 μg of DNA purified from cells was diluted to 0.5 ml with TE buffer, treated with ultrasound for 30 s (Bronson sonifier, equipped with a microtip), precipitated with ethanol, washed once with 70% ethanol, dried, and dissolved in 10 μl of TE buffer. Before immunoprecipitation (IP), DNA samples were boiled for 5 min, and chilled on ice. 0.5 μg of DNA was used in one IP reaction. MeDIP was done in 96-well plates as described (32 (link),33 (link)). Monoclonal antibodies to 5mC, 0.3 μg per IP reaction, were used. Mock IP was done without added antibodies. qPCR analysis of precipitated DNA was done with gene-specific primers as described above. 10% of the amount of input DNA used in IPs was analyzed in parallel by qPCR to estimate efficiency of IP. PCR reactions were run in triplicate. Standard dilutions of genomic DNA were included in each PCR run. Sequences of primers used are in Supplemental Table S1.

Immunofluorescence staining was performed in four-chamber microplates (ThermoFisher Scientific) according to previously established protocols (25 (link), 28 (link), 33 (link)). The primary and secondary antibody set included unconjugated mouse anti-5-methylcytosine monoclonal antibody (AMM99021, Aviva Systems Biology) at 1 mg/ml and Alexa488-linked donkey anti-mouse IgG (A-21202, ThermoFisher Scientific) at 5 mg/ml final concentrations. The cells were subsequently delineated with the cytoplasmic marker Cell Mask Red (ThermoFischer Scientific) and cell nuclei counterstained with DAPI. The specificity/dynamic range of the anti-5mC antibody was tested as previously reported in (29 (link)) (data not shown in here). Formalin-fixed tissue sections at 5 µm thickness were kept floating in 10% formalin at 2−8°C until immunofluorescence staining. Prior to staining, tissues were transferred to microwell plates, washed in PBS at room temperature, then stained as floating tissues using the same protocol that was applied to fixed cells.