Bisulfite-Seq analysis of foreskin melanocyte from skin03 (A20920)
Bisulfite-Seq analysis of foreskin melanocyte from skin03 (A20920) using Illumina HiSeq 2000
Refer to document 'Bisulfite Library Construction for Illumina Sequencing' from BC at the Roadmap Epigenomics Project site, Experimental Protocols page (URL: http://www.roadmapepigenomics.org/protocols/type/experimental/)
EXPERIMENT_TYPE
DNA Methylation
EXTRACTION_PROTOCOL
Refer to document 'Bisulfite Library Construction for Illumina Sequencing' from BC at the Roadmap Epigenomics Project site, Experimental Protocols page (URL: http://www.roadmapepigenomics.org/protocols/type/experimental/)
EXTRACTION_PROTOCOL_TYPE_OF_SONICATOR
Covaris-E210
EXTRACTION_PROTOCOL_SONICATION_CYCLES
0.92 min
DNA_PREPARATION_INITIAL_DNA_QNTY
1.00 ug
DNA_PREPARATION_FRAGMENT_SIZE_RANGE
300 bp
DNA_PREPARATION_ADAPTOR_SEQUENCE
Methylated Illumina PE adaptor
DNA_PREPARATION_ADAPTOR_LIGATION_PROTOCOL
Refer to document 'Bisulfite Library Construction for Illumina Sequencing' from BC at the Roadmap Epigenomics Project site, Experimental Protocols page (URL: http://www.roadmapepigenomics.org/protocols/type/experimental/)
DNA_PREPARATION_POST-LIGATION_FRAGMENT_SIZE_SELECTION
215-370 bp
BISULFITE_CONVERSION_PROTOCOL
2 rounds of Qiagen Epitect conversion
BISULFITE_CONVERSION_PERCENT
96.38
LIBRARY_GENERATION_PCR_TEMPLATE_CONC
N/A
LIBRARY_GENERATION_PCR_POLYMERASE_TYPE
Agilent PfuTurbo Cx Hotstart DNA Polymerase
LIBRARY_GENERATION_PCR_THERMOCYCLING_PROGRAM
2 minutes at 95C, 30 seconds at 98C, 15 cycles of 10 seconds at 98C, 30 seconds at 65C and 30 seconds at 72C, then 5 minutes at 72C
LIBRARY_GENERATION_PCR_NUMBER_CYCLES
5
LIBRARY_GENERATION_PCR_F_PRIMER_SEQUENCE
PE primer 1.0
LIBRARY_GENERATION_PCR_R_PRIMER_SEQUENCE
PE primer 2.0
LIBRARY_GENERATION_PCR_PRIMER_CONC
0.5 uM
LIBRARY_GENERATION_PCR_PRODUCT_ISOLATION_PROTOCOL
8% Novex TBE PAGE gel purification