Single-cell sequencing using 10X Genomics

Single cell samples were prepared using Single Cell 3^/ Library & Gel Bead Kit v2 and Chip Kit (10X Genomics) according to the manufacturer’s protocol. Briefly samples were FACS sorted using DAPI to select live cells followed by resuspension in 0.04% BSA-PBS. Nearly 1,200 cells/µl were added to each well of the chip with a target cell recovery estimate of 8,000 cells. Thereafter Gel bead-in Emulsions (GEMs) were generated using GemCode Single-Cell Instrument. GEMs were reverse transcribed, droplets were broken and single stranded cDNA was isolated. cDNAs were cleaned up with DynaBeads and amplified. Finally, cDNAs were ligated with adapters, post-ligation products were amplified, cleaned up with SPRIselect. Purified libraries were submitted to UCLA Technology Center for Genomics & Bioinformatics for quality check and sequencing. The quality and concentration of the purified libraries were evaluated by High Sensitivity D5000 DNA chip (Agilent) and sequencing was performed on NextSeq500.