title: Whole Genome Sequencing of U87MG Glioblastoma Cell Line by SOLiD
design: Long-Mate-Paired Library Construction: The U87MG genomic DNA 2x 50bp long mate-paired library construction was carried out using the reagents and protocol provided by Applied Biosystems (SOLiD 3 System Library Preparation Guide).  A similar protocol was reported previously [17]. Briefly, 45ug of genomic DNA was fragmented by HydroShear (Digilab Genomic Solutions Inc) to 1.0-2.5kb. The fragmented DNA was repaired by the End-It DNA End-Repair Kit (Epicentre). Subsequently, the LMP CAP adaptor was ligated to the ends. DNA Fragments between 1.2-1.7kb were selected by 1.0 % agarose gel to avoid concatamers and circularized with a biotinylated internal adaptor. Non-circularized DNA fragments were eliminated by Plasmid-Safe ATP-Dependent DNase (Epicentre) and 3ug of circularized DNA was recovered after purification. Original DNA nicks at the LMP CAP oligo/genomic insert border were translated into the target genomic DNA about 100bp by nick translation using E. coli DNA polymerase I. Fragments containing the target genomic DNA and adaptors were cleaved from the circularized DNA by single-strand specific S1 nuclease. P1 and P2 adaptors were ligated to the fragments and the ligated mixture was used to create two separate libraries with 10 cycles of PCR amplification. Finally, 250-300bp fragments were selected to generate mate paired sequencing libraries with average target genomic DNA on each end around 90bp by excision from PAGE gel and use as emulsion PCR template. Templated Beads Preparation: The templated beads preparation was performed using the reagents and protocol from the manufacturer (Applied Biosystems SOLiD 3 Templated Beads Preparation Guide).  SOLiD 3 Sequencing: The 2x50b mate-paired sequencing was performed exactly according to the Applied Biosystems SOLiD 3 System Instrument Operation Guide and using the reagents from Applied Biosystems.