EXPERIMENT_TYPE
DNA Methylation
EXTRACTION_PROTOCOL
Qiagen DNeasy mini kit, performed as per manufacturer's instructions
EXTRACTION_PROTOCOL_TYPE_OF_SONICATOR
Covaris S2
EXTRACTION_PROTOCOL_SONICATION_CYCLES
Standard fragment express, 6 cycles
DNA_PREPARATION_INITIAL_DNA_QNTY
5 µg
DNA_PREPARATION_FRAGMENT_SIZE_RANGE
100-150
DNA_PREPARATION_ADAPTOR_SEQUENCE
A: 5' P-GATCGGAAGAGCTCGTATGCCGTCTTCTGCTTG, B: 5' ACACTCTTTCCCTACACGACGCTCTTCCGATCT
DNA_PREPARATION_ADAPTOR_LIGATION_PROTOCOL
16degC for 16 hours with T4 DNA ligase (New England Biolabs)
DNA_PREPARATION_POST-LIGATION_FRAGMENT_SIZE_SELECTION
Two rounds of purification with AMPure XP beads (Agencourt)
BISULFITE_CONVERSION_PROTOCOL
Invitrogen MethylCode
BISULFITE_CONVERSION_PERCENT
99.5% of cytosines converted based on shotgun sequencing of unmethylated lambda phage control spiked into original genomic DNA sample
LIBRARY_GENERATION_PCR_TEMPLATE_CONC
>The adapter-ligated, bisulfite converted DNA was used in a 50 µl PCR reaction
LIBRARY_GENERATION_PCR_POLYMERASE_TYPE
Stratagene Pfu Turbo Cx
LIBRARY_GENERATION_PCR_THERMOCYCLING_PROGRAM
95degC 2 min; 98degC 30 sec, 4 cycles of 98degC 15 sec, 60degC 30 sec, 72degC 4 min; 72degC 10 min
LIBRARY_GENERATION_PCR_NUMBER_CYCLES
8
LIBRARY_GENERATION_PCR_F_PRIMER_SEQUENCE
5' AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT
LIBRARY_GENERATION_PCR_R_PRIMER_SEQUENCE
5' CAAGCAGAAGACGGCATACGAGCTCTTCCGATCT
LIBRARY_GENERATION_PCR_PRIMER_CONC
25 µM
LIBRARY_GENERATION_PCR_PRODUCT_ISOLATION_PROTOCOL
Two rounds of purification with AMPure XP beads (Agencourt)
GEO Accession
GSM983651