EXPERIMENT_TYPE: DNA Methylation
EXTRACTION_PROTOCOL:
Qiagen DNeasy mini kit, performed as per manufactu (show full text...)(hide...)Qiagen DNeasy mini kit, performed as per manufacturer's instructions'
EXTRACTION_PROTOCOL_TYPE_OF_SONICATOR: Diagenode Bioruptor
EXTRACTION_PROTOCOL_SONICATION_CYCLES:
20 cycles of: 30 seconds on high power, 2 minutes (show full text...)(hide...)20 cycles of: 30 seconds on high power, 2 minutes off
DNA_PREPARATION_INITIAL_DNA_QNTY: 5 µg
DNA_PREPARATION_FRAGMENT_SIZE_RANGE: 50-500 bp
DNA_PREPARATION_ADAPTOR_SEQUENCE:
A: 5' P-GATCGGAAGAGCTCGTATGCCGTCTTCTGCTTG, B: 5' A (show full text...)(hide...)A: 5' P-GATCGGAAGAGCTCGTATGCCGTCTTCTGCTTG, B: 5' ACACTCTTTCCCTACACGACGCTCTTCCGATCT
DNA_PREPARATION_ADAPTOR_LIGATION_PROTOCOL:
Standard Illumina genomic DNA library preparation (show full text...)(hide...)Standard Illumina genomic DNA library preparation ligation protocol (15 minutes at room temperature), except the adapters contained methylcytosine and were supplied by Illumina
DNA_PREPARATION_POST-LIGATION_FRAGMENT_SIZE_SELECTION:
Separation by electrophoresis on a 2% agarose gel (show full text...)(hide...)Separation by electrophoresis on a 2% agarose gel followed by excision of 140-210 bp fragments
BISULFITE_CONVERSION_PROTOCOL:
Standard Human Genetic Signatures MethylEasy Xceed (show full text...)(hide...)Standard Human Genetic Signatures MethylEasy Xceed bisulfite conversion kit protocol
BISULFITE_CONVERSION_PERCENT:
99.6% of cytosines converted based on shotgun sequ (show full text...)(hide...)99.6% of cytosines converted based on shotgun sequencing of unmethylated lambda phage control spiked into original genomic DNA sample
LIBRARY_GENERATION_PCR_TEMPLATE_CONC:
One third of the 140-210 nt adapter-ligated, bisul (show full text...)(hide...)One third of the 140-210 nt adapter-ligated, bisulfite converted DNA was used in each 50 µl PCR reaction
LIBRARY_GENERATION_PCR_POLYMERASE_TYPE: Stratagene Pfu Turbo Cx
LIBRARY_GENERATION_PCR_THERMOCYCLING_PROGRAM:
95°C 2 min; 98°C 30 sec, 4 cycles of 98°C 15 sec, (show full text...)(hide...)95°C 2 min; 98°C 30 sec, 4 cycles of 98°C 15 sec, 60°C 30 sec, 72°C 4 min; 72°C 10 min
LIBRARY_GENERATION_PCR_NUMBER_CYCLES: 4
LIBRARY_GENERATION_PCR_F_PRIMER_SEQUENCE:
5' AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGC (show full text...)(hide...)5' AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT
LIBRARY_GENERATION_PCR_R_PRIMER_SEQUENCE: 5' CAAGCAGAAGACGGCATACGAGCTCTTCCGATCT
LIBRARY_GENERATION_PCR_PRIMER_CONC: 25 µM
LIBRARY_GENERATION_PCR_PRODUCT_ISOLATION_PROTOCOL:
Separation by electrophoresis on a 2% agarose gel (show full text...)(hide...)Separation by electrophoresis on a 2% agarose gel followed by excision of 180-300 bp fragments.
GEO Accession: GSM432691