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: 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' A (show full text...)(hide...)A: 5' P-GATCGGAAGAGCTCGTATGCCGTCTTCTGCTTG, B: 5' ACACTCTTTCCCTACACGACGCTCTTCCGATCT
DNA_PREPARATION_ADAPTOR_LIGATION_PROTOCOL:
16C for 16 hours with T4 DNA ligase (New England B (show full text...)(hide...)16C 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 (A (show full text...)(hide...)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 sequ (show full text...)(hide...)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 (show full text...)(hide...)>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:
95C 2 min; 98C 30 sec, 4 cycles of 98C 15 sec, 60C (show full text...)(hide...)95C 2 min; 98C 30 sec, 4 cycles of 98C 15 sec, 60C 30 sec, 72C 4 min; 72C 10 min
LIBRARY_GENERATION_PCR_NUMBER_CYCLES: 8
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:
Two rounds of purification with AMPure XP beads (A (show full text...)(hide...)Two rounds of purification with AMPure XP beads (Agencourt)
GEO Accession: GSM1010983