title: single cell seq of pdac endothelial tissue from mus musculus
design: To construct the RNA Library, we used the Single Cell RNA v1.1 kit by Scale Biosciences, which employs 3-level combinatorial indexing. Fixed cells were distributed across a 96- well RT Barcode Plate (20,000 cells per well) for cDNA synthesis and incorporation of 96 unique RT barcodes. Because endothelial cells account for approximately 60-70% of non myocytes in the heart, we sought to reduce their representation in order to avoid oversampling, as previously described. To achieve this, one column of the plate was allocated to endothelial cells, six columns to cardiomyocytes, and five columns to leukocytes and mesenchymal cells. Following indexed reverse transcription, cells were pooled by centrifugation and redistributed into a 384-well Ligation Barcode Plate, where ligation adaptors containing the TruSeq Read 1 sequence and 384 unique ligation barcodes were added. Cells were then pooled and diluted to a concentration of 400 cells/l and distributed across a 96-well plate at 1600 cells per well for second strand synthesis, followed by an enzymatic cleanup step to breakdown cells. This was followed by tagmentation to incorporate the Nextera Read 2 sequence for subsequent amplification of the mRNA transcript. Indexed PCR was then carried out to add 96 unique index i5 Barcodes along with four distinct i7 indices for improve nucleotide diversity and color balancing during sequencing. The final RNA library was purified using SPRIselect beads, and quality was assessed on an Agilent TapeStation with High Sensitivity D5000 ScreenTape. Library concentration was quantified by Qubit fluorometry and qPCR prior to sequencing. Sequencing was performed on an Illumina NovaSeq 6000 platform at a final loading concentration of 200 pM, targeting a depth of 20,000 read pairs per cell.