After washing and purification, the ChIP samples were analyzed by real-time qPCR using primers with the sequences listed in Supplementary Table S1.
aProtein ORFs used were predicted from the sequences listed under cDNA from Table 1.
DNA sequencing was performed on a random sample from each subpopulation using the sequencing primers listed in Table 1, with a total number of 18 samples sequenced (S (n=1), RS (n=7), I (n=2), R (n=3) and HR (n=5)).
Both strands of purified PCR products were directly sequenced in 10 μL reactions using the sequencing primers listed in Additional file 1. Cycle sequencing was conducted using dRhodamine Dye Terminator reagents and a PE-ABI 377 automated DNA sequencer (Perkin Elmer – Applied Biosystems).
Transcript levels of osteogenic marker genes (ALP, COL1A1, SPP1/OPN, and RUNX2), adipogenic marker genes (FABP4/αP2, CEBPA/C/EBP-α, LEP/Leptin, and PPARG2/PPAR-γ2), and GAPDH (an internal standard) were evaluated using the primer sequences listed in Table 1.
Clone alignments were performed using the GAP4 assembly program, version 6 [ 40], using the sequences of the GenBank versions listed in the legend of Fig. 1 and Additional file 1.
Secondly, the MSA was built using the sequences obtained through BLAST search, and the results were used to update the original sequence list, which was further refined using HMMER.
These are obtained from the data series GSE38046, using the Sequence Read Accession (SRA) numbers listed below.
The ligated RNAs were reverse transcribed and PCR amplified using the oligo primer sequences listed in Table S1.
PCR products were cloned into pCR-Blunt II-TOPO (Invitrogen) and analyzed using the listed sequencing primers (Table S1-a and -b).
