disease state: CML clinical phase: at diagnosis (Dx) therapy-response: NA cell type: CML Bone Marrow HSC cell subtype: Lin-CD34+CD38-
Treatment protocol
Single cell suspension of cells were FACS sorted to obtain HSC cells
Extracted molecule
total RNA
Extraction protocol
Cells were sorted into 96 well plates with 4µl lysis buffer (Neuclease free water, RNAsout (Invitrogen), 0.1M DTT, 10mM dNTP, 10% NP40) and immediately frozen at -80°C.
Label
FAM-MGB
Label protocol
cDNA synthesis and amplification was performed by adding a mix of 96 Taqman assays with a final concentration of 0.025x and Taq/SSIII enzyme reaction mix (CellsDirect One-Step RT-qPCR kit, Invitrogen) and PCR cycling was performed as follows, cDNA synthesis: 60min at 50oC and 2 min at 95oC, amplification: 15sec at 95oC and 4 min at 60oC repeated 25 times. The cDNA was diluted 5 times with water prior to qPCR on the BioMark HD (Fluidigm Corporation, San Francisco, CA). All primers were used at 12.5x in the final reaction and Taqman universal master mix (Applied Biosystems, Foster City, CA) was used for detection of the qPCR products. The qPCR was performed using the 96.96 Dynamic Array Integrated Fluidic Circuits (IFC) (Fluidigm Corporation, San Francisco, CA) chip in combination with the BioMark HD with the following thermal cycler program: thermal mix 50°C for 2 minutes followed by 70°C for 30 minutes and 25°C for 10 minutes, UNG at 50°C for 2 minutes, hot start 96.5°C for 10 minutes, PCR cycle 40x (96°C 15 seconds, 60°C for 60 seconds).
Hybridization protocol
n/a
Scan protocol
n/a
Description
CML_Plate2_PatNr.7_Dx All patients included in this dataset responded well to TKI, but leukemic cells (Ph+) can still be detected in patient bone marrow.
Data processing
Ct values calculated and exported using Fluidigm Biomark software. Data normalized by subtraction of the median Ct per cell followed by z-score scaling.
