GSM5060608: NT_41_NEG_2_; Homo sapiens; RNA-Seq
Sorted cells were counted, and adjusted to a target of 5,000 cells in a 38 µL volume of 1X PBS (Gibco) and 0.04% BSA (Sigma-Aldrich). Cells, reverse transcription (RT) master mix (10X Genomics), primer-coated 10X gel beads, and partitioning oil were loaded onto a Single Cell A chip (10X Genomics) and into a 10X Genomics Chromium Controller instrument for microfluidic droplet-based encapsulation of cells, RT reagents, and gel beads. RT reaction was performed within droplets in a thermocycler (Bio-Rad) and following the 10X Genomics incubation protocol. Droplets were then broken down, and the released barcoded cDNAs were purified using magnetic beads, and amplified by PCR. cDNA concentrations were determine using a Qubit 3.0 fluorometer and Qubit HS dsDNA assay kit (both, ThermoFisher). Enzymatic digestion and size selection were performed to generate the optimal cDNA size for library construction. Sequencing primers P5, P7, R2 and sample-specific index primers were added during library construction. Quality and size of generated libraries was verified using the Bioanalyzer high-sensitivity DNA analysis kit (Agilent). Library concentrations were determined using kapa DNA library quantification kit (KAPA Biosystems). Libraries were sequenced on an Illumina HiSeq 4000