title: GSM2060412: Cell93-B11 cKit+ Flt3ITD/ITD,Dnmt3afl/- MxCre  AML-2; Mus musculus; RNA-Seq
library_construction_protocol: Single CD117+ (c-Kit+) cells were prepared using the C1™ Single-Cell Auto Prep System (Fluidigm, San Fransisco, CA), according to the manufacturer’s instructions.  In short, sorted cells were counted and resuspended at a concentration of 333,333 cells per 1 mL DPBS then loaded onto a primed C1 Single-Cell Auto Prep Integrated Fluidic Chip for mRNA-Seq (5-10 µm). After the fluidic step, cell separation was visually scored to identify wells containing single live cells. In two independent rounds, 52 and 56 cells were captured. Cells were lysed on the chip and reverse transcription was performed using the Clontech SMARTer® Kit with the mRNA Seq: RT + Amp (1771x) according to the manual. After the reverse transcriptase step, cDNAs were transferred to a 96 well plate and diluted with 6 µl C1™ DNA Dilution Reagent. cDNAs were quantified using the Quant-iT™ PicoGreen® dsDNA Assay Kit (Life Technologies, Grand Island, NY) and Agilent High Sensitivity DNA Kit (Agilent Technologies (Santa Clara, CA). Libraries for the first 48 visually validated cells from each of the two independent captures were prepared using the Nextera XT DNA Library Preparation Kit (Illumina Inc, Santa Clara, CA).  In each single-cell library preparation, a total of 125pg cDNA was tagmented at 55 °C for 20 minutes. Next, libraries were pooled and purified on the AMPure® bead-based magnetic separation Kit prior to a final quality control check using both the Qubit® dsDNA HS Assay Kit (Life Tecnologies, Grand Island, NY) and Agilent High Sensitivity DNA Kit. The majority of cDNA fragments were confirmed to be between 200-602 bp, qualifying the libraries for sequencing. All 96 single-cell RNA-Seq libraries were sequenced on one HiSeq 2500 gel.
attributes.tag: GEO Accession
attributes.value: GSM2060412