GSM9194555: asynchronous; Homo sapiens; RNA-Seq
Trypsinised cells were prepared as a single cell suspension. Dissociated cells were mixed with DropSeq barcoded beads and droplet generation oil to generate droplets containing single cells and beads. This was performed using syringes with set flow rates on a PDMS co-flow microfluidic droplet generation chip (FlowJem). Droplet captures were performed for 7 mins to obtain 0.5 mL of beads, with ~2.5% of beads associated with a cell. Cells were captured using the microfluidic devices described for Dropseq following the Online Drop-seq Laboratory Protocol version 3.1 (www.mccarrolllab.com/dropseq). Following droplet lysis, beads with barcoded RNAs were retrotranscribed, treated with exonuclease I, and amplified by PCR. Pooled cDNAs were then tagmented with the Nextera XT DNA Library kit (Illumina Cat# FC-131-1024) and custom primers. The tagmented and multiplexed cDNA libraries were sequenced in Nextseq 500 using Nextseq500 High Output v.2 kit (75 cycles, Illumina Cat# FC-404-2005) following Macosko et al. recommendations and additional modifications described in PMID 33852842.