GSM5008880: PtenS398A/S398A; Mus musculus; RNA-Seq
Before sorting, a small-sized (5-10 μm) C1 Single-Cell AutoPrep IFC microfluidic chip (Fluidigm) was primed using the script “SMART-Seq v4: Prime Rev B (1771x) for 5-10 μm diameter cells” according to the manufacturer's instruction. 2.5 μL of Cell Suspension-Viability reagent, prepared  by mixing Cell Suspension buffer (Fluidigm) and LIVE/DEAD Viability/Cytotoxicity Kit for mammalian cells (Thermo Fisher) components at a ratio of 1:1:50 (Ethidium homodimer-1 : Calcein AM : Cell Suspension buffer) was added in the cell loading inlet of the C1 chip. Cells were sorted directly onto the cell loading inlet of the C1 chip, using optimized single-cell sorting parameters on a BD FACSARIA instrument (BD Biosciences). Typically, cells were sorted at an approximate speed of 3500 events/second with low-pressure settings, yielding approximately 1000 LT-HSCs (~0.2% of the total number of events), in a volume of 4.5-5 µL, in around 10 minutes. After sorting, the cell loading inlet of the C1 chip contained approximately 7.5 μL of sorted LT-HSCs at a final concentration of ~130-140 cells/μL and cell buoyancy ratio of ~60-65% (murine primary HSCs : Cell Suspension-Viability reagent). The C1 chip was then processed on a Fluidigm C1 instrument (Fluidigm) using the script “SMART-Seq v4: Cell Load Rev B (1771x) for 5-10 μm diameter cells”. This protocol was designed to capture individual LT-HSC in up to 96 capture chambers in the C1 chip in approximately 30 minutes. The chip was then transferred to an EVOS FL Cell Imaging System (Thermo Fisher), and each cell was imaged under bright-field and fluorescence illumination to visualize the viability stain. C1 reagents (Fluidigm) and SMART-Seq v4 Ultra Low Input RNA Kit for the Fluidigm C1 System (Clontech) were added to the designated wells according to the manufacturers' instructions. The chip was then returned to the Fluidigm C1 system and the script “SMART-Seq v4: Sample Prep Rev B (1771x), for 5-10 um diameter cells” was executed, which took ~8 h and included single cell lysis, reverse transcription, and full-length cDNA synthesis. After completion of the protocol, the amplified single cell cDNAs were harvested in a total of 8 μL C1 DNA Dilution Reagent (Fluidigm). 1 μL of the diluted cDNA was analyzed using Agilent High Sensitivity DNA Kit on an Agilent BioAnalyzer 2100 (Agilent Technologies, Inc.) and quantified using Quant-iT PicoGreen dsDNA Assay kit (Thermo Fisher) on a SpectraMax M3 Microplate Reader (Molecular Devices). The average size of the single cell cDNA synthesized was between 1300 to 1,700 bp in length and the typical yield was ~400-1200 pg/μL. Single cell cDNA libraries were constructed using Nextera XT DNA Library Kit (Illumina) according to the modified protocols described in the SMART-Seq V4 Ultra Low Input RNA Kit for the Fluidigm C1 System, IFCs User Manual (Clontech). 100-300 pg of each amplified single cell cDNA was used as the input for Nextera XT library construct, and Illumina N7 and S5 indexes were added (Illumina) for tagmentation followed by PCR amplification using an Applied Biosystems Veriti 96-Well Thermal Cycler (Thermo Fisher). cDNA libraries were pooled and purified with Agencourt AMPure XP beads (Beckman Coulter, Inc.) twice. Final cDNA libraries sizes were validated using the Agilent Bioanalyzer 2100 and quantified by quantitative PCR using KAPA SYBR FAST qPCR kit (Kapa Biosystems, Inc.) on a CFX96 Real-Time system (BioRad).  The libraries were then sequenced on the NextSeq 500 system (Illumina, Inc.) using a pair-end 75 cycle sequencing run to achieve one million reads per cell.