GSM6890722: hDE_rep2; Homo sapiens; RNA-Seq
Cells were washed once with PBS and lifted using TryplE Following the Drop-seq Laboratory Protocol version 3.1 (http://mccarrolllab.com/dropseq/),  sorted single cell suspensions were resuspended at 1x105 cells per mL in PBS + 0.01% BSA. The diluted cell suspension, barcoded Oligo-dT beads (Chemgenes, MACOSKO-2011-10), and droplet generation oil (Biorad, 1864006) was run through a PDMS co-flow microfluidic droplet generation device (Nanoshift, custom built based on Macosko et al. datafile 1 [Macosko et al., 2015] at flow rates of 4,000 μL per hour, 4,000 μL per hour, and 15,000 μL per hour, respectively. Droplet breakage, bead isolation, and cDNA synthesis were performed as described (Macosko et al., 2015). cDNA libraries were tagmented with Nextera XT DNA Library Preparation kit (Illumina, FC-131-1024) and sequencing libraries were amplified and individually barcoded. Agencourt AMPure XP beads (Beckman Coulter, A63881) were used for purification of cDNA and sequencing libraries according to the manufacturer's instructions. Final sequencing libraries were analyzed on an Agilent High Sensitivity DNA bioanalyzer chip to determine average basepair size and final library concentrations were determined with a Qubit High Sensitivity DNA assay kit (Invitrogen, Q32854).  Drop-seq libraries were sequenced at paired-end (20-50) on a Nextseq500 using a Nextseq500/550 High Output v2 75-cycle kit (Illumina, FC-404-2005).