NextSeq 500 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 3 ul of Smart-seq3 lysis buffer supplemented with 0.025 pg of 5'-molecular spike-in and lysate was used for library preparation without nucleic acid extraction. Smart-seq3 library preparations were performed as published: https://www.protocols.io/view/smart-seq3-protocol-bcq4ivywLibrary and with following modifications: The cDNA was cleaned up after reverse transcription. For the pre-amplification PCR different amounts of fwd primer were chosen (0.1, 0.5, 1.0 uM) in presence or absence of template switching oligo.