Labelling was performed under the following conditions 94°C for 5 minutes, 25 cycles of 93°C for 30 sec, 50°C for 30 sec 72°C for 30 sec and 1 cycle of 72°C for 5 min. Probes were denatured at 99°C for 5 min and snap chilled before hybridisation.
One of 96 indexed primers (8mer_SC_TC 1 to 96 - ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNBBBBNNNNXXXXXXXXCGTTTTTTTTTTTTTTVN - generic primer where X represents an 8 base index as described in Additional file 1, N is A, C, G or T, B is C, G or T and V is A, C or G) was added to each RNA sample (1 μl of 10 μM), then heated to 70 °C and snap chilled on ice.
Sample plate was heated at 95 °C for 5 min, snap chilled and loaded into autosampler of the instrument.
The 6-FAM-labeled PCR products were diluted tenfold in water, and 0.5 μL was mixed with 9.5 μL of HiDi formamide (Applied Biosystems, Inc., USA) and 0.25 μL of GeneScan Liz 250 internal size standards (Applied Biosystems, Inc., USA) and heated to 95°C for 2 minutes snap chill down on ice.
Aliquots of 20 µl each were removed at 10-, 20-, 30-, 40-, 50-, 60-, 90-, 120-, and 150-s time intervals, and the reaction was stopped by snap-chilling the samples in liquid nitrogen.
To one half 1.5 µM of [3H] AdoMet was added and aliquots of 20 µl were withdrawn at different time intervals (at 15-, 30-, 45-, 60-, 120-, 180-, 240-, and 300-s time intervals) and the reactions were stopped by snap chilling the samples in liquid nitrogen.
This was followed by snap chilling the samples for 10 minutes.
The dried sample was resuspended in nuclease free water and mixed with hybridization mix containing blocking solution (Agilent Technologies, part number 5190-0456) and Hi-RPM hybridization buffer (Agilent Technologies, part number 5190-0456) and incubated at 100°C for 5 minutes followed by snap chill on ice for 5 minutes.
The dried sample were resuspended in nuclease free water and mixed with Hybridization Mix containing blocking solution and Hi-RPM Hybridization Buffer and incubated at 100 °C for 5 min followed by snap chill immediate cooling on ice for 5 min. The samples were hybridized on the Human_miRNA_ version 3.15 × 8 array.
100 μl sheared chromatin aliquots were then placed on 95°C heat block for 10 min. This was followed by snap chilling the samples for 10 min. The samples were then diluted, precleared and processed further in a manner identical to the ChIP protocol described in the previous section.
First, denaturing of 10 μg of aaRNA together with 6 μg of random primers (Life Technologies) was carried out by incubation at 70°C for 10 min and snap-chill on ice.
