Agilent 4200 tapestation high sensitivity d5000 screentape

Libraries were prepared following the 10x Genomics Single Cell 3′ protocol. Single cells were dissociated, washed and resuspended in a 1x PBS/0.04% BSA solution at a concentration of 1000 cells/μl to remove dead cells and contaminants. A Cellometer (Nexcelom) was used to determine cell viability and cells were normalized to 1 × 10⁶ cells/mL. Cells were then combined with a master mix including reverse transcription reagents. With this, gel beads carrying the Illumina TruSeq Read 1 sequencing primer, a 16bp 10x barcode, a 12bp unique molecular identifier (UMI) and a poly-dT primer were loaded onto the chip, together with oil for the emulsion reaction. Reverse transcription occurs in nanoliter-scale gel beads in emulsion (GEMs) so that all cDNAs within a GEM share a common barcode. After this reverse transcription reaction, the GEMs were broken and full length cDNA purified with Silane Dynabeads and SPRI beads then assayed on an Agilent 4200 TapeStation High Sensitivity D5000 ScreenTape (Santa Clara, CA) for qualitative and quantitative analysis. Illumina P5 and P7 sequences (San Diego, CA), a sample index and TruSeq read 2 primer sequences were added via End Repair, A-tailing, Adaptor Ligation and PCR. Sequences were generated using paired end sequencing on an Illumina sequencing platform at a minimum of 50,000 reads/cell.