Instrument: Illumina HiSeq 2500
Strategy: RNA-Seq
Source: TRANSCRIPTOMIC
Selection: cDNA
Layout: SINGLE
Construction protocol: Cells were sorted directly into TriZOL on a FACS Aria III and frozen at -80C. Gen-Elute linear polyacrylamide (Sigma) was added to the RNA in TRIzol, followed by the addition of 0.2 volumes of chloroform and vortexing. After centrifuging at 14,000xg for 5 minutes, the aqueous phase was transferred to a clean tube. Nucleic acid was precipitated with isopropanol and 3M sodium acetate pH 5.5 (Ambion) at -20°C overnight and then centrifuged at maximum speed for 30 minutes at 4°C. The supernatant was discarded and the pellet was washed by adding 0.5mL of 100% ethanol, centrifuging for 15 minutes at 4°C, and discarding the supernatant. The nucleic acid pellet was allowed to dry and was resuspended in 5 μL of molecular grade water. RNA-DGE derived from the Single Cell RNA Barcoding and Sequencing (SCRB-Seq) strategy 3’ Digital Gene Expression (3’ DGE)