title: Whole genome shotgun bisulfite sequencing of the H1 mesendoderm cell line
design: Five µg of genomic DNA was extracted from frozen cell pellets using the DNeasy Mini Kit (Qiagen, Valencia, CA) and spiked with 25 ng unmethylated cl857 Sam7 Lambda DNA (Promega, Madison, WI). The DNA was fragmented with a Covaris S2 (Covaris, Woburn, MA) to 100-150 bp, followed by end repair and addition of a 3' A base. Cytosine-methylated adapters provided by Illumina (Illumina, San Diego, CA) were ligated to the sonicated DNA at 16C for 16 hours with T4 DNA ligase (New England Biolabs). Adapter-ligated DNA was isolated by two rounds of purification with AMPure XP beads (Beckman Coulter Genomics, Danvers, MA). Adapter-ligated DNA (450 ng) was subjected to sodium bisulfite conversion using the MethylCode kit (Life Technologies, Carlsbad, CA) as per manufacturer's instructions. The bisulfite-converted, adapter-ligated DNA molecules were enriched by 4 cycles of PCR with the following reaction composition: 2.5 U of uracil-insensitive PfuTurboCx Hotstart DNA polymerase (Stratagene), 5 µl 10X PfuTurbo reaction buffer, 31 µM dNTPs, 1 µl Primer 1, 1 µl Primer 2 (50 µl final). The thermocycling parameters were: 95C 2 min, 98C 30 sec, then 4-8 cycles of 98C 15 sec, 60C 30 sec and 72C 4 min, ending with one 72C 10 min step. The reaction products were purified using AMPure XP beads (two rounds). Up to three separate PCR reactions were performed on subsets of the adapter-ligated, bisulfite-converted DNA, yielding up to three independent libraries from the same biological sample.
attributes.tag: EXPERIMENT_TYPE
attributes.value: DNA Methylation
attributes.tag: EXTRACTION_PROTOCOL
attributes.value: Qiagen DNeasy mini kit, performed as per manufacturer's instructions
attributes.tag: EXTRACTION_PROTOCOL_TYPE_OF_SONICATOR
attributes.value: Covaris S2
attributes.tag: EXTRACTION_PROTOCOL_SONICATION_CYCLES
attributes.value: Standard fragment express, 6 cycles
attributes.tag: DNA_PREPARATION_INITIAL_DNA_QNTY
attributes.value: 5 µg
attributes.tag: DNA_PREPARATION_FRAGMENT_SIZE_RANGE
attributes.value: 100-150
attributes.tag: DNA_PREPARATION_ADAPTOR_SEQUENCE
attributes.value: A: 5' P-GATCGGAAGAGCTCGTATGCCGTCTTCTGCTTG, B: 5' ACACTCTTTCCCTACACGACGCTCTTCCGATCT
attributes.tag: DNA_PREPARATION_ADAPTOR_LIGATION_PROTOCOL
attributes.value: 16°C for 16 hours with T4 DNA ligase (New England Biolabs)
attributes.tag: DNA_PREPARATION_POST-LIGATION_FRAGMENT_SIZE_SELECTION
attributes.value: Two rounds of purification with AMPure XP beads (Agencourt)
attributes.tag: BISULFITE_CONVERSION_PROTOCOL
attributes.value: Invitrogen MethylCode
attributes.tag: BISULFITE_CONVERSION_PERCENT
attributes.value: 99.5% of cytosines converted based on shotgun sequencing of unmethylated lambda phage control spiked into original genomic DNA sample
attributes.tag: LIBRARY_GENERATION_PCR_TEMPLATE_CONC
attributes.value: One third of the adapter-ligated, bisulfite converted DNA was used in each of three 50 µl PCR reaction
attributes.tag: LIBRARY_GENERATION_PCR_POLYMERASE_TYPE
attributes.value: Stratagene Pfu Turbo Cx
attributes.tag: LIBRARY_GENERATION_PCR_THERMOCYCLING_PROGRAM
attributes.value: 95C 2 min; 98C 30 sec, 4 cycles of 98C 15 sec, 60C 30 sec, 72C 4 min; 72C 10 min
attributes.tag: LIBRARY_GENERATION_PCR_NUMBER_CYCLES
attributes.value: 4
attributes.tag: LIBRARY_GENERATION_PCR_F_PRIMER_SEQUENCE
attributes.value: 5' AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT
attributes.tag: LIBRARY_GENERATION_PCR_R_PRIMER_SEQUENCE
attributes.value: 5' CAAGCAGAAGACGGCATACGAGCTCTTCCGATCT
attributes.tag: LIBRARY_GENERATION_PCR_PRIMER_CONC
attributes.value: 25 µM
attributes.tag: LIBRARY_GENERATION_PCR_PRODUCT_ISOLATION_PROTOCOL
attributes.value: Two rounds of purification with AMPure XP beads (Agencourt)
attributes.tag: GEO Accession
attributes.value: GSM818003