Supplementary Figure 2: Modular ligation strategy for streamlined production of barcoded nucleosomal 601 DNA.
From: Accelerated chromatin biochemistry using DNA-barcoded nucleosome libraries
(a) DNA ligation using non-palindromic overhangs for the preparation of barcoded nucleosomal 601 DNA (‘BC-601’). Complementary single-stranded DNA spanning nucleotides (nt) 10-30 of the forward Ion Torrent Adaptor A (‘Adaptor A10-30’) and the respective 6 bp nucleosome (NUC) barcodes (‘BC-NUC’) were annealed to afford ‘BC’. Note that an AA overhang was introduced at the 3’-end of the BC bottom strand to prevent blunt end self-ligation. Additionally, a 5’ overhang of 4 bases was incorporated into the bottom strand for the subsequent ligation reaction, in which 1.1 eq. of the BC was combined with 1.0 eq. of a BsaI-DraIII-digested nucleosomal 601 sequence (‘601’). The mixture was in situ phosphorylated using T4 DNA PNK and ligated with T4 DNA ligase. The respective sequences are shown at the bottom. (b) Analysis of starting material (601 and BC, left) and reaction products (right) by native gel electrophoresis followed by ethidium bromide (EtBr) DNA staining revealed quantitative ligation. For the identity of the NUC barcodes, see Supplementary Table 1. H: A/T/C.
