XP11
Harvested cells at the required densities were combined with gel beads containing the barcoded information along with a mixture of cells and enzymes. Oil surfactant droplets in a microfluidic "double-cross" system were used for encapsulation to generate Gel Beads-In-Emulsions (GEMs). GEMs were passed through a reservoir and collected while the gel beads were lysed to release the barcode sequence. The cDNA fragment was then reverse transcribed before the sample was labeled. The gel beads and the oil droplets are ruptured and PCR amplification was performed using the generated cDNA as a template. The products of all GEMs were mixed and a standard sequencing library is constructed. The cDNA was first enzymatically digested into fragments of about 200  300 base pairs (bp), together with the library building process of traditional second-generation sequencing such as sequencing junction and primers. Finally, the DNA library was generated by PCR amplification. Cell capture was performed using the official library kit (10X Genomics Chromium Single-Cell 3' kit, V3) according to manufacturers instructions. Following the capture of 10,000 target cells, sequencing was performed using the NovaSeq6000 sequencing platform (paired-end multiplexing run, 150 bp) by LC-Bio Technology Co. Ltd. (Hangzhou, China). A sequencing depth of 50,000 reads per cell was required.
SUB15423025
XP11