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. Cells were FACS sorted and lysed into 5 ul of Phusion HF (1/500 diluted) buffer supplemented with 0.025 pg of 3'-molecular spike-in and lysate was used for library preparation without nucleic acid extraction. Libraries were prepared in 3 variations of the SCRB-seq protocol. (1) standard protocol as described by Soumillon et al., 2014 (2) as (1) but omitting the ExonucleaseI step prior to cDNA amplification and (3) PCR master-mix was added to each well individually, omitting both the pooling and Exonuclease I steps.