NextSeq 500 paired end sequencing; Whole exome sequencing of human MDS/AML OCI-M2 and patient-derived bone marrow cell lines treated with 5-azacytidine
Whole exome sequencing of human MDS/AML OCI-M2 and patient-derived bone marrow cell lines treated with 5-azacytidine
Genomic DNAs for whole exome sequencing (WES) were isolated from cultured 5-Azacytidine-sensitive (AZA-S) cells (derived from ancestral OCI-M2 line), 5-Azacytidine-resistant (AZA-R) subclones and hematopoietic cells from bone marrow aspirates obtained from patients diagnosed with Myelodysplastic syndrome that were collected in accordance with the patient's informed consent. OCI-M2 AZA-R subclones evolved from AZA-S cells after treatment with bi-weekly 8 M AZA doses exceeding IC50 for duration of 6 weeks. After their establishment the AZA-R cell lines were maintained by weekly 2 M doses of 5-azacytidine in IMDM + 20%FBS. AZA-S cells were cultivated in IMDM + 20%FBS without 5-Azacytidine. Genomic DNAs were isolated with DNeasy Blood & Tissue Kit (Qiagen) from a starting material of approximately 5 x 106 nucleated bone marrow cells and cell lines. Genomic DNA libraries were prepared with Roche/NimbleGen SeqCap EZ Library Kit (KAPA HyperCap Workflow v3.0) (Indianapolis, IN) covering 47 Mb of all medically relevant genes (~2000).