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Bisulfite-Seq analysis of WGBS_Lib 61 derived from human adult CD3 cells
WGBS REMC Sequencing on Illumina
Project News:
2013/7/15: EXPERIMENT XML file 'BI.experiment.G1828.2013-07-12.xml' uploaded
alias: WGBS_Lib 61
center_name: BI expected_number_runs: 3 STUDY_REF: G1828 SAMPLE_DESCRIPTOR: BioSam 1236 LIBRARY LIBRARY_NAME: WGBS_Lib 61 LIBRARY_STRATEGY: Bisulfite-Seq LIBRARY_SOURCE: GENOMIC LIBRARY_SELECTION: RANDOM LIBRARY_LAYOUT: SINGLE POOLING_STRATEGY: none LIBRARY_CONSTRUCTION_PROTOCOL: 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. SPOT NUMBER_OF_READS_PER_SPOT: READ_INDEX: 0 READ_CLASS: Application Read READ_TYPE: Forward BASE_COORD: 1 PLATFORM ILLUMINA INSTRUMENT_MODEL: Illumina HiSeq 2000 SEQUENCE_LENGTH: 99 BASE CALLS GAPipeline RTA1.15.19.5 QUALITY SCORES GAPipeline RTA1.15.19.5 ATTRIBUTES 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) |
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