Genomic DNA (1.5~5µg) was fragmented to 100-500 bp using a Covaris S2. Purified DNA fragments were end-repaired. After A-tailing, the DNA fragments were ligated with methylated paired-end adapters. Adapter-attached DNA fragments of 300-400 bp, which contain 150-250 bp genomic DNA inserts, were gel-purified. The purified DNA fragments were subjected to two cycles of sodium bisulfite conversion using the EpiTect Bisulfite kit (Qiagen). Adapter-attached, bisulfite-converted DNA molecules were enriched by PCR using PfuTurboCx Hotstart DNA polymerase (Stratagene). The PCR amplified DNA fragments were subjected to a second gel size selection to remove PCR primers and adapter dimers. The enriched library was quantified using a Qubit fluorometer and Quant-iT dsDNA HS Assay Kit. Library sequencing was performed using 101 BP PE Illumina technology.
BISULFITE_CONVERSION_PROTOCOL
2x5hrs Epitect Kit
DNA_PREPARATION_INITIAL_DNA_QNTY
5 ug
EXTRACTION_PROTOCOL_SONICATION_CYCLES
Covaris shearing, duty cycle 5%, intensity 5, cycles / burst 200, duration 9 minutes
DNA_PREPARATION_ADAPTOR_LIGATION_PROTOCOL
T4 Ligase (2,000U/ul) 16C o/n
DNA_PREPARATION_ADAPTOR_SEQUENCE
Illumina paired end adapter
LIBRARY_GENERATION_PCR_R_PRIMER_SEQUENCE
PE1.0
DNA_PREPARATION_POST-LIGATION_FRAGMENT_SIZE_SELECTION
250-400
BISULFITE_CONVERSION_PERCENT
99.5%
EXTRACTION_PROTOCOL
Standard Protocol (Smith et al., Methods 48, 226-232)
LIBRARY_GENERATION_PCR_PRIMER_CONC
25uM
DNA_PREPARATION_FRAGMENT_SIZE_RANGE
130-280
EXPERIMENT_TYPE
DNA Methylation
LIBRARY_GENERATION_PCR_THERMOCYCLING_PROGRAM
Smith et al., Methods 48, 226-232
LIBRARY_GENERATION_PCR_PRODUCT_ISOLATION_PROTOCOL
SPRI Beads
LIBRARY_GENERATION_PCR_F_PRIMER_SEQUENCE
PE2.0
LIBRARY_GENERATION_PCR_TEMPLATE_CONC
NA
LIBRARY_GENERATION_PCR_POLYMERASE_TYPE
Pfu Turbo Cx hot start
LIBRARY_GENERATION_PCR_NUMBER_CYCLES
5-8
EXTRACTION_PROTOCOL_TYPE_OF_SONICATOR
Covaris S2 (TM)
GEO Accession
GSM1172595