title: Bisulfite-Seq analysis of WGBS_Lib 16 derived from human Mobilized CD34 cells
design: 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.
attributes.tag: BISULFITE_CONVERSION_PROTOCOL
attributes.value: 2x5hrs Epitect Kit
attributes.tag: DNA_PREPARATION_INITIAL_DNA_QNTY
attributes.value: 5 ug
attributes.tag: EXTRACTION_PROTOCOL_SONICATION_CYCLES
attributes.value: Covaris shearing, duty cycle 5%, intensity 5, cycles / burst 200, duration 9 minutes
attributes.tag: DNA_PREPARATION_ADAPTOR_LIGATION_PROTOCOL
attributes.value: T4 Ligase (2,000U/ul) 16C o/n
attributes.tag: DNA_PREPARATION_ADAPTOR_SEQUENCE
attributes.value: Illumina paired end adapter
attributes.tag: LIBRARY_GENERATION_PCR_R_PRIMER_SEQUENCE
attributes.value: PE1.0
attributes.tag: DNA_PREPARATION_POST-LIGATION_FRAGMENT_SIZE_SELECTION
attributes.value: 220-380
attributes.tag: BISULFITE_CONVERSION_PERCENT
attributes.value: 99.5%
attributes.tag: EXTRACTION_PROTOCOL
attributes.value: Standard Protocol (Smith et al., Methods 48, 226-232)
attributes.tag: LIBRARY_GENERATION_PCR_PRIMER_CONC
attributes.value: 25uM
attributes.tag: DNA_PREPARATION_FRAGMENT_SIZE_RANGE
attributes.value: 160-340
attributes.tag: EXPERIMENT_TYPE
attributes.value: DNA Methylation
attributes.tag: LIBRARY_GENERATION_PCR_THERMOCYCLING_PROGRAM
attributes.value: Smith et al., Methods 48, 226-232
attributes.tag: LIBRARY_GENERATION_PCR_PRODUCT_ISOLATION_PROTOCOL
attributes.value: SPRI Beads
attributes.tag: LIBRARY_GENERATION_PCR_F_PRIMER_SEQUENCE
attributes.value: PE2.0
attributes.tag: LIBRARY_GENERATION_PCR_TEMPLATE_CONC
attributes.value: NA
attributes.tag: LIBRARY_GENERATION_PCR_POLYMERASE_TYPE
attributes.value: Pfu Turbo Cx hot start
attributes.tag: LIBRARY_GENERATION_PCR_NUMBER_CYCLES
attributes.value: 5-8
attributes.tag: EXTRACTION_PROTOCOL_TYPE_OF_SONICATOR
attributes.value: Covaris S2 (TM)
attributes.tag: GEO Accession
attributes.value: GSM916052