Gel extraction protocol

Each library was individually hybridized to the biotinylated probes in the presence of RNA blocking oligos complementary to the Illumina adapters for 48 hours at 62 °C. For each 38 μL final volume reaction, 15 μL of ancient DNA library (80–270 ng) was heated for 5 min at 95 °C, incubated for 5 min at 62 °C, and mixed with ca. 4 μg of each pool of blocking oligos (N500 or N700), prewarmed (62 °C) 2× hybridization buffer (10× SSPE, 10 mM EDTA and 0.2 % Tween-20), and ca. 500 ng prewarmed (2 min at 62 °C) mix of biotinylated RNA. After incubation, the hybridization mix was added to 500 ng (50 μL) M-280 streptavidin Dynabeads (Invitrogen), that had been washed three times and resuspended in 162 μL 1 M NaCl, 10 mM Tris-HCl, pH 7.5, 1 mM EDTA, and 1× Denhardt’s solution. After 40 min at room temperature, the beads were pulled down and washed once at room temperature for 15 min with 0.2 mL 1× SSC/0.1 % Tween-20, followed by three 10 min washes at 62 °C with 0.2 mL 0.1× SSC/0.1 % Tween-20, resuspending the beads once at each washing step. Hybrid-selected DNA was eluted twice with 50 μL 0.1 N NaOH. After 10 min at 20 °C, the beads were pulled down and the supernatant transferred to a tube containing 75 μL 1 M Tris-HCl, pH 7.5. The captured single stranded DNA fragments were then purified using the Qiagen Gel extraction protocol. DNA was eluted with 30 μL of EB buffer.

PCR primer pairs (forward: 5'-GCTGTGGGTGAAAATGCCTT-3' and reverse:
5'-TGAAGGGACATTGGAGATTG-3') were used to amplify the region containing the breakpoint
between exon 44 and exon 51 caused by a deletion in the DMD gene.
Each PCR reaction contained AmpliTaq Gold^® 360 PCR Master
Mix (Applied Biosystems), 10 μM primers and 50-100 ng of template gDNA/μL in a final
volume of 25 μL. The cycling conditions were: 95°C 10 min, 35 cycles of 95 °C for 15
s, 62 °C for 3 s and 72 °C for 60 s, and a final extension at 72 °C for 7 min. The
quality of the amplified products was assessed using agarose gel electrophoresis and
the PCR fragment was extracted from the gel using a Qiagen Gel extraction protocol
(QIAquick^® gel extraction kit; Qiagen, Valencia, CA). Clean PCR product
was used for Sanger sequencing in a 3500 Series Genetic Analyzer.