Large construct capture (LCC) of human osteosarcoma: NAAHDI Normal
"BACs and Fosmids tiling the genomic regions of RB1 (RP11-1001I7, chr13:48869794-49059184), TP53 (G248P86004B11, chr17:7564399-7602817), CDKN2A (RP11-478M20, chr9:21947304-22110179), ATRX (RP11-1145J4, chrX:76738001-76906419; RP11-42M11, chrX:76829558-76972747; RP11-125C15, chrX:76926270-77082337), SATB2 (RP11-644D20, chr2:200125368-200293969; RP11-989H13, chr2:200175383-200351494), PTEN (RP11-383D9, chr10:89557022-89719607; RP11-846G17, chr10:89676236-89853112), NF1 (CTD-2583F5, chr17:29391131-29600804; RP11-876L22, chr17:29551639-29740620), and NF2 (CTD-2335H16, chr22:29967139-30072498; RP11-551L12, chr22:30038549-30179920) were grown and DNA isolated using the Large-Construct Kit (12462, Qiagen). DNAs were quantified and combined in equimolar representation using Droplet Digital PCR (ddPCR) and primers (BACbone Forward: 5'-AGCTCCTGGTCACGTTCATT-3' and BACbone Reverse: 5'-CTGTATGTGGTCGGGATGTG-3') targeting the vector backbone of the genomic constructs. DNA baits produced by Klenow labeling using biotinylated random hexamer (IDT) was bound to MyOne Streptavidin C1 Dynabeads (65001, Invitrogen) and used to capture Nextera genomic libraries in a rotisserie using conditions previously described (Maricic et al., 2010) with modifications. The hybridization was additionally blocked with Nextera library blockers and captured library recovered by PCR directly on the washed beads using custom primers (InPE 1.0: 5'-AATGATACGGCGACCACC-3', InPE 2.0: 5'-GTTCGTCTTCTGCCGTAT-3'). "
NCI-phs000468
L061616_TARGET_NAAHDI_10A_LCC