GSM2690986: ATAC-seq for patient CLL10 before Ibrutinib treatment; Homo sapiens; ATAC-seq
Heparinized peripheral blood was obtained from CLL patients after signed informed consent. Peripheral blood mononuclear cells (PBMCs) were isolated using Ficoll-Hypaque (GE Healthcare, Uppsala, Sweden) centrifugation. Patients were screened for CLL characteristic chromosomal aberrations including deletions on 13q14, 11q22, and 17p13 and for trisomy 12 by FISH analysis. The IGHV and TP53 mutational status was determined by sequencing (LGC Genomics, Berlin, DE). PBMCs from CLL patients were cryopreserved in RPMI 1640 supplemented with 40% FCS and 10 % DMSO. ~10^5 cells were pelleted by centrifuging for 5 min at 4 °C at 300 x g. After centrifugation, the pellet was carefully resuspended in the transposase reaction mix (12.5 ml 2xTD buffer, 2 ml TDE1 (Illumina) and 10.25 ml nuclease-free water, 0.25 µl 5% Digitonin (Sigma)) for 30 min at 37 °C. Following DNA purification with the MinElute kit eluting in 11 µl, 1 µl of the eluted DNA was used in a quantitative PCR (qPCR) reaction to estimate the optimum number of amplification cycles. Library amplification was followed by SPRI size selection to exclude fragments larger than 1,200 bp. DNA concentration was measured with a Qubit fluorometer (Life Technologies). Library amplification was performed using custom Nextera primers. The libraries were sequenced by the Biomedical Sequencing Facility at CeMM using the Illumina HiSeq3000/4000 platform and the 25-bp paired-end configuration.