RRBS of homo sapiens: peripheral blood chronic lymphocytic leukemia cells: Sample DFCI-5340-T-02
Peripheral blood mononuclear cells from all patients were isolated by Ficoll-Hypaque density gradient centrifugation, cryopreserved with 10% dimethyl sulfoxide, and stored in vapor-phase liquid nitrogen until time of use. Tumor cells were purified by flow cytometric isolation of CD19+/CD5+ cells for downstream nucleic acid extraction. Reduced representation bisulfite sequencing (RRBS) libraries were generated from 25 to 100 ng of input DNA using the Ovation Methyl-Seq System (NuGen) following the manufacturer's recommendations. These reads were aligned to the human hg19 genome using BSmap with flags -v 0.05 -s 16 -w 100 -S 1 -p 8 u. An average of 23.1 million reads per sample was aligned correctly. An average of 5.4 million CpGs was covered per sample. The methylation state of each CpG was determined by comparing bisulfite-treated reads aligning to that CpG with the genomic reference sequence. The extent of methylation was computed by dividing the number of observed methylated cytosines (which did not undergo bisulfite conversion) by the total number of reads aligned to that CpG.
NCI-phs001998
DFCI_5340_Post-HSCT