5'-chloroacetamido-5'-deoxythymidine

1 μg of total RNA preparation was used in a first strand cDNA synthesis reaction (10 μL final volume) using Superscript III and oligo(dT)20 (Invitrogen) following the manufacturer's instructions. After cDNA amplification, the 10 μL reaction was diluted with 60 μL of nuclease-free water, 5 μL of which was used in a 20 μL real time qRT-PCR reaction using SYBR Green Jumpstart Taq Ready Mix (Sigma-Aldrich, Cat.# S4438) and a Roche Lightcycler 480II instrument. Expression of the MADS-box transcription factor FLOWERING LOCUS C (FLC, At5g10140) was normalised to UBIQUITIN CONJUGATING ENZYME1 (UBC, At1g14400) using the comparative Cq (quantification cycle) method [13 (link)-16 ]. Primers were designed to bridge exons: 5'-AGC CAA GAA GAC CGA ACT CA-3' and 5'-TTT GTC CAG CAG GTG ACA TC-3' for FLC; 5'-CTG CGA CTC AGG GAA TCT TCT AA-3' and 5'-TTG TGC CAT TGA ATT GAA CCC-3' for UBC.

ChIP assays were performed using a kit following the manufacturer’s instructions (#17-295; Millipore). Accordingly, cells were first treated with 1% formaldehyde and incubated at 37°C for 10 min. Next, cells were washed twice with ice-cold PBS containing protease inhibitors. Cells were then scraped and pelleted by centrifugation at 2,000 RPM for 4 min at 4°C. Then, cells were resuspended in SDS Lysis Buffer (Millipore, #20-163) and incubated for 10 min on ice. After samples were centrifuged for 10 min at 13,000 RPM at 4°C, the supernatants were diluted 10 times by adding ChIP Dilution Buffer (#20-153; Millipore) containing protease inhibitors. The diluted supernatants were then treated with 75 μl of a 50% slurry of Protein-A Agarose/Salmon Sperm DNA (#16-157C; Millipore) at 4°C for 30 min with agitation. After centrifugation, supernatants were immunoprecipitated with antibodies against Smad4, Smad2, Smad3, Prdm16, GAPDH or isotype-matched control IgG and 60 μl of a 50% slurry of Protein-A Agarose/Salmon Sperm DNA at 4°C for 1 h with rotation. Agarose was pelleted using centrifugation at 1,000 RPM for 1 min at 4°C. The pellets were washed for 5 min in Low Salt Immune Complex Wash Buffer (#20-154; Millipore) once, High Salt Immune Complex Wash Buffer (#20-155; Millipore) once, LiCl Immune Complex Wash Buffer (#20-156; Millipore) once, and TE Buffer (#20-157; Millipore) twice. To amplify DNA bound to the immunoprecipitates, elution buffer (1% SDS, 0.1 M NaHCO₃) was added to each sample followed by agitation and incubation for 15 min with rotation at room temperature. Eluates were then mixed with NaCl (final concentration of 0.2 M) and incubated for 4 h at 65°C followed by adding EDTA (0.01 M), Tris-HCl, pH 6.5 (0.04 M), and Proteinase K (0.04 mg/ml). Samples were then incubated for 1 h at 45°C, and DNA was recovered using phenol/chloroform extraction coupled with ethanol precipitation. Pellets were washed with 70% ethanol and air-dried. Lastly, pellets were resuspended in an appropriate buffer for PCR, and PCR products were analyzed on a 2% agarose gel. The immunoprecipitated DNA was also analyzed by qPCR using locus specific primers and normalized to input DNA. Relative fold enrichment in each locus was quantified relative to the control as described above (qRT-PCR) as well as in our published studies (Parajuli et al., 2018 (link); Zhang et al., 2015 (link)). The following primers were used: PRDM16-For 5′-CAT​CTC​CCC​AGC​ATT​GTC​AGT-3′; PRDM16-Rev 5′-GGA​GCG​CCG​AAC​ACG​GAA​TG-3′; JUNB-For 5′-GGC​AAA​GCC​CAG​GGT​CAA​TA-3′; JUNB-Rev 5′-AAA​GCT​AGT​AAG​CGG​CCT​GG-3′; GAPDH-For 5′-CGG​GAT​TGT​CTG​CCC​TAA​TTA​T-3′; GAPDH-Rev 5′-GCA​CGG​AAG​GTC​ACG​ATG​T-3′.

Prdm16-For 5′-TCC​CAC​CAG​ACT​TCG​AGC​TA-3′; Prdm16-Rev 5′-AAA​GTC​GGC​CTC​CTT​CAG​TG-3′; Gapdh-For 5′-CAC​CAT​CTT​CCA​GGA​GCG​AG-3′; Gapdh-Rev 5′-CAC​CAT​CTT​CCA​GGA​GCG​AG-3′.