High Throughput bisulfite sequencing of sample 14838, library 2
DNA libraries from each tissue sample were prepared using a customized DNA sample preparation protocol. Sample genomic DNA (5 µg) was fragmented using a S2 ultrasonicator (Covaris, Woburn, MA). Sizing of 50-100 ng fragmented DNA was performed using an Experion automated electrophoresis system and Experion 1K DNA Analysis kit (Bio-Rad, Hercules, CA) to confirm the correct average size and size range expected for each application. Fragmented DNA was repaired to blunt ends using the END-It kit (Epicentre Biotechnologies, Madison, WI). The end-repaired DNA was purified and recovered using a Qiagen MinElute PCR purification column. Addition of an ‘A’ base to the 3’ end of blunt DNA was then accomplished using Klenow exo (3’- >5’ exonuclease minus) (New England Biolabs, Ipswich, MA). The A-tailed DNA was then purified using the Qiagen MinElute PCR purification columns. Adapters with a 3’ ‘T’ overhang were ligated to the end-modified DNA (see below).  For whole genome bisulfite sequencing, modified Illumina paired-end (PE) adapters were used in which cytosine bases in the adapter are replaced with 5-methylcytosine bases (Integrated DNA Technologies, Coralville, IA) to prevent conversion of the adapter sequence during bisulfite treatment. Double stranded annealed adapters were prepared by annealing and then ligated to the end-repaired DNA samples using ultrapure, rapid T4 ligase (Enzymatics, Beverly, MA) and purified on a Qiagen PCR purification column.  DNA libraries were bisulfite converted with the Zymo EZ DNA Methylation kit (Zymo Research, Orange, CA) according to the manufacturer’s specifications. We then performed a series of MethyLight-based, quality control steps to ensure complete bisulfite conversion as described in the report from Campan and colleagues (PMID: 18987824). Specifically, each bisulfite-converted DNA sample was subject to four MethyLight assays to measure bisulfite conversion completeness and the amount of bisulfite converted DNA from each sample.   Quantitative PCR (qPCR) was performed on a fraction (1 ul) of the Adapter-ligated library using primers from Illumina (PE 1.0/2.0), master mix (Applied Biosystems 2X SYBR Green) and a serial dilution curve of PhiX library DNA as a standard using an ABI 7900HT real-time PCR machine (Applied Biosystems, Foster City, CA).  Based on the amplification curve from the qPCR, the optimal cycle number was determined for each library as plateau cycle, where the reaction exits the linear phase of amplification, minus 3 cycles.  Amplification of the Adapter-ligated library was performed using Pfu Cx Turbo Taq Polymerase (Stratagene, Carlsbad, CA) according to manufacturers instructions and Illumina primers (PE 1.0/2.0). A total of 30% of the bisulfite converted adapter-ligated library was added to a total of 450 ul of reaction mix, vortexed repeatedly, then divided into 9 separate wells (50uL each) for thermal cycling.  Following PCR the 9 wells are pooled and PCR products are purified using a QIAquick PCR Purification kit (Qiagen, Valencia, CA). Size fractionation and removal of adapter dimers was performed using 10% TBE acrylamide gels and a 25bp ladder size standard (Invitrogen, Carlsbad, CA). An acrylamide gel slice containing the target library (typically 200-300bp) was excised from the gel, crushed and sheared to break up the acrylamide, soaked in a Tris/Ammonium Acetate buffer (10mM/2.5mM) and precipitated with ethanol.  Cluster density was estimated using qPCR with both PhiX library serial dilutions and other libraries previously run with known cluster density. Amplified library DNA was denatured with NaOH prior to cluster generation. The denatured DNA was diluted to a 4pM concentration. 
USC-EC
tumor.AMA502A1_lib2