Evercode WT Mega v2.2.1 v1
Authors: Biosciences Parse , Rebboah Elisabeth 
Published: January 2024

This protocol describes the original Parse Biosciences Evercode WT Mega v2 protocol for single-nucleus or single-cell RNA-seq of 1,000,000 nominal nuclei or cells. Unlike other scRNA-seq methods that physically separate individual cells into different compartments to label transcripts with cell-specific barcodes, Evercode WT Mega uses the cells (or nuclei) themselves as “containers” in which intracellular transcripts are labeled using combinatorial indexing. In practice, cells are split into different wells, a well-specific barcode is appended to intracellular transcripts, and cells are then pooled back together. Repeating this process several times ensures a high likelihood that each cell travels through a unique combination of wells. Consequently, the transcriptome of each individual cell is labeled with a unique combination of well-specific barcodes. Unlike previous methods that scale linearly with the number of available compartments and barcodes, this method scales exponentially with the number of barcoding rounds, enabling a massive increase in the number of cells that can be sequenced, while minimizing doublets. The products of this protocol are up to 16 subpools of varying size, where each molecule has a cell/nucleus barcode and UMI, ready for Illumina short-read sequencing. The cell/nucleus barcode is unique within each subpool, so the subpol index acts as the fourth barcode to uniquely barcode all the cells/nuclei within an experiment. Please see the attachment for the original Parse Biosciences protocol. The main deviations from the original protocol are 1. loading 15 ul rather than 14 ul in the Round 1 barcoding plate and 2. saving all "leftover" barcoded nuclei or cells at -80C for potential repeats. The first part of the protocol, Section 1, describes barcoding cells or nuclei. Briefly, fixed cells/nuclei are thawed and added to the Round 1 reverse transcription barcoding plate at 37,500 cells/nuclei per well across 96 wells. Individual samples from each tissue are distributed in the sample barcoding plate with at least 1 well per sample. Within the fixed cells/nuclei, RNA is reverse transcribed using oligodT and random hexamer primers and the first barcode is annealed. After RT, cells/nuclei are pooled and distributed in 96 wells of the Round 2 ligation barcoding plate for in situ barcode ligation. After Round 2, cells/nuclei are pooled and redistributed into 96 wells of the Round 3 ligation barcoding plate for barcode 3 and Illumina adapter ligation. Finally, cells/nuclei are counted using a hemocytometer, distributed into up to 16 subpools, and lysed. The second part, Section 2, describes template switching and amplification of the full-length cDNA. Importantly, full-length cDNA from Section 2 is the input for cDNA Exome Capture. For non-exome captured Illumina libraries, proceed to Section 3: Preparing Libraries for Sequencing. Section 3 describes Illumina library preparation with 100 ng of full-length cDNA per subpool. Subpool cDNA is fragmented and Illumina P5/P7 adapters are ligated during the final amplification. Importantly, if using single indices (WX100) please follow Appendix D: Single Indexing. If using dual indices (WX200) follow main protocol. Libraries with 5% PhiX spike-in are sequenced on an Illumina sequencer as paired-end, single-index (140/86/6/0) or dual-index reads (130/86/8/8).