Direct Library Preparation (DLP)-Based Single Cell Total RNAseq (DLP-scRNAseq)

Cell spotting was performed using the cellenONE (Cellenion) platform as previously described for the Direct Library Preparation Plus (DLP+) single-cell genome sequencing protocol (Laks et al., 2019). For single cells, the upstream RNAseq preparation steps including cell lysis, RNA fragmentation, cDNA synthesis and adapter addition were performed as described above for the SMARTer bulk protocol, but generally with volumes in nanoliters as opposed to microliters. The step-by-step details of the protocol are attached in Supplementary Text File (pages 5–29). For optimal spotting of reaction mixes other than the lysis/fragmentation mix, 0.05% Tween-20 was spiked into the reactions. Reaction mixes and primers were filtered using spin-x columns (Cat. No. CLS8162; Merck) whenever spotting proved to be problematic. All steps up to and including the introduction of cell-specific indices during the first round of PCR (pre-rRNA depletion), were performed in nanoliter volumes using Takara Smart Chips (Takara Bio Inc). These arrays consist of a 72 × 72 (5184) well layout each of which able to hold a volume of approximately 100 nl. After 5 cycles of the first round of PCR, the chip was inverted and spun down to pool all reactions into one tube. Subsequent steps were performed according to the SMARTer Stranded Total RNAseq Kit v2–Pico Input Mammalian manufacturer’s instructions.

The SMARTer^® kit comes with indexing primers that allow the barcoding of a maximum of 96 samples. To increase the number of cells that could be processed, we designed our own barcodes based on the following requirements: (1) the random primer and strand-switching oligos were to be anchored to Illumina sequencing primer sequences, and (2) primers used for the first round of PCR must have complementary sequences to the Illumina sequencing primer anchors internally, followed by indices in the middle, and P5/P7 priming sites at their distal ends. We thus designed 72 × 72 dual indexing primers enabling 5184 unique cell-specific barcodes (Supplementary Table 1).