title: Pooling-based genetic mapping of DNA methylation: Sample 130723_PINKERTON_0257_AC28G4ACXX_L7_CGATGT
design: We pooled genomic DNA derived from 60 Yoruba HapMap cell lines (parental samples from 30 HapMap trios, purchased from Coriell).  48 µg of this DNA was spiked with 240 ng unmethylated cl857 Sam7 Lambda DNA (Promega, Madison, WI) to yield 0.5% W/W lambda DNA.  The DNA was fragmented with a Covaris instrument (Covaris) in 50 uL volumes (duty factor 10%, peak incident power 175, 200 cycles per burst, 40 seconds duration, 5.5-6.0C), followed by end repair, adenylation, and adapter ligation using the Truseq DNA LT Sample Prep Kit (Illumina) as per manufacturer’s instructions.  Purification steps were performed using Agencourt Ampure beads (Beckman-Coulter).  All 24 indexed methylated adapters from Truseq DNA LT Sample Prep Kit Sets A and B (Illumina) were used to construct the libraries.  Adapter-ligated DNA of 400-500 bp was isolated by 2% agarose gel electrophoresis using low range ultra agarose (Bio-Rad) with Sybr gold Nucleic acid gel stain (Invitrogen), and fractions were purified using MinElute Gel Extraction Kit (Qiagen).  Two of the 24 libraries were reserved and not bisulfite-converted for control purposes.  Sodium bisulfite conversion was carried out on the 22 remaining libraries using the Epitect Bisulfite Kit (Qiagen #59104) as per manufacturer’s 2006 instructions, except the reaction mix incubation cycle from the Whole-Genome Bisulfite Sequencing for Methylation Analysis was used (Illumina, Part # 15021861 Rev. A) and consisted of 95 °C for 5 min, 60 °C for 25 min, 95 °C for 5 min, 60 °C for 85 min., 95 °C for 5 min., 60 °C for 175 min., 95 °C for 5 min., 60 °C for 180 min., ending with a 20 °C hold.  Bisulfite-converted products were purified using MinElute PCR Purification Kit (Qiagen).  Adapter-ligated, bisulfite-converted libraries were enriched using KAPA HiFi HotStart Uracil+ ReadyMix uracil-insensitive polyermase (D-mark Biosciences #KK2801).  Thermocycler parameters for bisulfite-converted libraries consisted of 98 °C for 45 sec; four cycles of 98 °C for 15 sec., 65 °C for 30 sec., 72 °C for 30 sec., ending with a 4 °C hold.  Adapter-ligated, non-bisulfite-converted control libraries were enriched using the reagents and protocol from the Truseq DNA LT Sample Prep Kit (Illumina).  Thermocycler parameters consisted of 98 °C for 45 sec.; four cycles of 98 °C for 10 sec., 60 °C for 30 sec., 72 °C for 30 sec., ending with a 4 °C hold.  PCR reaction products were purified using Agencourt Ampure beads (Beckman-Coulter).  Library validation was performed using the KAPA SYBR FAST Universal qPCR Library Quantification Kit (D-mark Biosciences, #KK4824) to measure the concentration of viable sequencing template molecules, as well as the Agilent Bioanalyzer High Sensitivity DNA Assay (Agilent) to determine the size and distribution of the template molecules.  Libraries were further concentrated using the MinElute PCR Purification Kit (Qiagen).  Each non-control library was divided across eight sequencing lanes in three flowcells, and each control library was divided across four of these sequencing lanes, which were in two of the flowcells.