DNBSEQ-G400 sequencing; Single-cell RNA sequencing with molecular spikes to determine RNA counting accuracy
Single-cell RNA sequencing with molecular spikes to determine RNA counting accuracy
HEK293FT cells were dissociated using TrypLE. Cell suspension was supplemented with 3'-molecular spikes and captured in a Chromium controller using the v2 kit as specified by the manufacturer. 10x Genomics libraries were prepared according to the v2 protocol with the following changes: 10 ul of GEM-RT mix was used and the remainder of the GEM-RT mix was discarded to reduce cell numbers. The  fragmentation step was extended to 10 minutes. The double-sided bead-cleanup after the fragmentation was changed to a ratio of 0.6:1 and 1:1 respectively.  Similarly, the post-ligation cleanup (step 3.4) was increased to 1:1 ratio instead of 0.8:1. The double-sided post-indexing PCR cleanup was performed at 0.6:1 and 1:1 ratios respectively.