Illumina HiSeq 2500 paired end sequencing; Identification of potential new therapeutic approaches for JAK2 V617F mutant patients
Identification of potential new therapeutic approaches for JAK2 V617F mutant patients
Use of human tissue was in compliance with the ethical and legal framework of the Human Tissue Act. Experiments had ethical approval from the NRES committee of the regional NHS health research authority (14/LO/0489 and 17/LO/0888). Primary JAK2 V617F positive samples were obtained from the Manchester Cancer Research Centre Biobank (HTA 30004) following authorisation by the Tissue Biobankâ€™s scientific sub-committee. Patient details are outlined in Table 1. The CD34+ cell population was enriched using CliniMACS (Miltenyi Biotec) according to standard protocols. CD34+ cells were isolated from the peripheral blood of seven patients with myelofibrosis. Cells were cultured for 24 hours in Fishers 20% HS, 10ng/ml IL-3 10ng/ml Flt3, 50ng/ml SCF with either 100nM CBL0137 or carrier control before extraction of RNA. Qiagen RNeasy Plus kit. TruSeq Stranded mRNA assay (Illumina)