synthesis at 16C with a T7-RA5 primer. The resulting double stranded cDNA underwent a final second IVT step at 37C for 14hrs to make aRNA. These two rounds of linearly amplified aRNA products now carried the 3′-end of the polyA transcripts for mapping to coding regions plus the sample barcode to indicate which PCP it came from, UMI sequence for counting unique cell-endogenous parent mRNA molecules and one of the flanking sequence (RA5 adapter) for Illumina sequencing. Second round aRNAs were fragmented chemically using NEBNext® Magnesium RNA Fragmentation Module (Cat#E6150S), column purified using RNA MinElute (Qiagen) for final Illumina cDNA library preparation steps.
