|Public on Jul 16, 2015
|reprogramming intermediates of hiF-T cells
|surface antigen enrichment: SSEA3-
treatment: DOX days 0-10
|Refer to the section Cell Culture and Reprogramming in the Supplemental Experimental Procedures of the related manuscript.
|hiF-T fibroblasts were expanded on gelatin-coated dishes in hiF medium composed of basal medium (DMEM-F12 Glutamax, 1X MEM-NEAA, 1X beta-ME, 0.2X P/S) supplemented with 10% ES-FBS and 16 ng/ml FGFbasic. HUES64 and hIPSC-T were expanded in feeder-free conditions using Essential 8 (E8) medium. The attachment matrix for PSC was either Matrigel (MTG) or Vitronectin (VTN) with no significant differences.
|Genomic DNA was extracted with the QIAamp DNA Mini Kit (QIAGEN)
Reduced Representation Bisulfite Sequencing (RRBS) Illumina libraries were prepared according to a standard gel-free pipeline(Boyle et al., 2012) starting from 10ng of genomic DNA.
|Illumina HiSeq 2500
|Reads were aligned on build hg19 of the human genome with MAQ(Li, Ruan, & Durbin, 2008) in bisulfite alignment mode.
The methylation level at single CpGs was calculated as the fraction of reads with unconverted cytosines over the total number of reads at that position. The methylation level for promoters was calculated as the mean of the methylation levels of the CpGs within the promoter weighted by the number of reads covering each CpG.
Differentially methylated promoters were defined as those exhibiting a methylation difference of 0.2 or greater between any two conditions and used for standard k-means clustering.
Supplementary_files_format_and_content: chrom, chromstart, chromend, 'number methylated cytosines/number total cytosines', score, strand
|Nov 18, 2014
|Last update date
|May 15, 2019
|Shannan J Ho Sui
|Harvard T.H. han Scholol of Public Health
|655 Huntington Ave, Building 2, Rm 437A
|Integrative analyses of human reprogramming reveal dynamic nature of induced pluripotency
|Integrative analyses of human reprogramming reveal dynamic nature of induced pluripotency [RRBS]