Klenow buffer

End-repair was performed by adding 45 μL of H₂O, T4 DNA ligase buffer with 10 mM ATP (NEB; 10 μL), dNTP mix (10 mM), T4 DNA polymerase (15 U; NEB), Klenow DNA polymerase (5 U; NEB), and T4 PNK (50 U; NEB) to the sample and incubating for 30 min at 20°C. A single base was added each to cDNA fragment by adding Klenow buffer (NEB), dATP (1 mM), and Klenow 3′ to 5′ exo- (15 U; NEB). The mixture was then incubated at 37°C for 30 min. Standard Illumina adapters (FC) were ligated to the cDNA fragments using 2× DNA ligase buffer (Enzymatics), 1 μL of adapter oligo mix and DNA ligase (5 U; Enzymatics). The sample was incubated at 25°C for 15 min. The sample was purified in a 2% low-melting point agarose gel, and a slice of gel containing 200-bp fragments was removed and the DNA purified. The polymerase chain reaction (PCR) was used to enrich the sequencing library. A 10 μL aliquot of purified cDNA library was amplified by PCR using the pfx DNA polymerase (2 U; Invitrogen) and with 1 μl of genomic primers 1.1 and 2.1 (Illumina). PCR cycling conditions included a denaturing step at 98°C for 30 sec, 12 cycles of 98°C for 10 sec, 65°C for 30 sec, 68°C for 30 sec, and a final extension at 68°C for 5 min. All libraries were sequenced on a HiSeq 2000 platform to a depth of over 190 million 50 bp reads using standard Illumina operating procedures.

Single-stranded purified oligonucleotides were annealed in a solution containing 0.125 M TRIS-HCl pH 7,4, NaCl 0.5 M. Annealing mixtures were heated to 95° C for 5 min and slowly chilled overnight to room temperature. About 100 ng of double-stranded oligonucleotide was 32P labeled with 5 U of Klenow fragment (3’–5’ exo-) (New England Biolabs, Beverly, MA) at 37°C for 1 h in 50 µl of a reaction mixture containing: 1× Klenow buffer (New England Biolabs), dTTP 1 mM, dGTP 1mM, 30 µCi [α32P]dATP (Amersham) and 30 µCi [α32P]dCTP (Amersham). Labeling mixtures were subsequently centrifuged through a G-25 Sephadex column to remove excess of unincorporated nucleotide. Gel retardation assays were performed by mixing 0.4 ng (about 5 × 105 cpm) of probe with 1 µl of in vitro synthesized Sp-Elk protein and 5 µg of poly(dI-dC)/poly(dI-dC) duplex in a buffer containing 20 mM HEPES (pH 7.9), 0.75 mM DTT, 57.5 mM KCl, 2.5 mM MgCl2, 0.05 mM EDTA and 10% glycerol. The binding reaction was performed on ice for 15 min and then the DNA-binding complexes were resolved by gel electrophoresis on an 8% polyacrylamide gel, 1× TBE with 300 V at 4° C. After 2–3 h run, gels were dried and exposed overnight to Biomax MS film. The sequences of oligonucleotides used are: Cy313F, 5’-GAGTATCAACAGGAAGTAGGT-3’ and Cy313R, 5’-ACCTACTTCCTGTTGATACTC-3’.