Subsequently 96 μl of DNA ligase in Ligation Dilution Buffer was added to the sample wells, resealed and incubated at 37 °C for 10 min to allow for the ligation reaction.
The 14 base pair dumbbell was converted into two precursor RNA strands by placing nicks in the single strand loop regions to allow for straightforward ligation by T4 RNA ligase 1 (for sequences see Supplementary file 1).
The remainder of the reaction was then diluted ≥threefold in 37°C buffer containing no ethylene glycol and 10 mM MgCl2 and incubated for 1 min to allow for DNA ligation.
Both the p2309-2 and pD2309-3 primers contained complementary sequences, containing a HindIII restriction site and a start and stop codon of the target gene to allow for efficient ligation of both flanking regions of the target gene by fusion PCR.
Similar to a protocol developed for low input DNA concentrations, this particular order was critical in this MeDIP-Seq protocol to allow for adapter ligation on double-stranded DNA as the conditions used in the immunoprecipitation step cause the DNA to become single-stranded.
To allow for robust ligation, we phosphorylated the "reverse" oligos of our adapters at the 5′ end (Materials and Methods, Figure S1); although this can increase the number of adapter dimers, these dimers are effectively removed by size selection as noted below.
II) RNA fragmentation by metal hydrolysis to allow for more efficient ligation of the 5′-end RNA adapter and better accessibility for the RNA loop adapter.
A KasI restriction site was added to the 3'-end of GFP, YFP and CFP to create a two-amino-acid linker (glycine-alanine) and to allow for in-frame ligation with the C-terminal ELP fusion tag.
This would allow for ligation of multiple Q-blocks in a single step.
