RNA-seq Pacbio MAS-seq 16-mer prepared with 10X 3', of homo sapiens: PBMCs
Cells were captured by adding 10 l of sample (2,000 cells/ul K562:3T3 cells; 1650 cells/ul Fresh PBMCs, 2000 cells/ul DSP/Methanol vs Fresh PBMCs, 3000cells/ul Multi-Fix PBMC) directly into Fluent PIP bead reagent mix (PIPseq T20 Single Cell RNA kit v4.0). For K562:3T3 samples, and fresh only PBMC experiments, 2 l of Recombinant RNase Inhibitor (Takara; cat no. 2313A) was also added. For fix vs fresh PBMC sample comparison, Protector RNase Inhibitor (Roche, 3335399001) was used. Cell capture, cell lysis, cDNA synthesis and whole transcriptome amplification (WTA) were performed as per the manufacturers instructions. Briefly, cells suspended in the emulsion were lysed and mRNA was captured on PIP-beads before breaking the emulsion prior to reverse transcription. cDNA underwent WTA, using 11 cycles for 3T3:K562 cDNA and 14 cycles for PBMC cDNAs, with 8 min extensions. Amplified cDNA was isolated from the PIPs then SPRI purified and quantified using Qubit. Library integrity assessment was performed on a high-sensitivity D5000 ScreenTape on 4150 Tapestation (Agilent Technologies).