Technical Bulletin For Single Cell Analysis MULTI-seq, multiplexing using lipid-tagged indices, is a novel tool that facilitates multiplex single-cell and single-nucleus RNA sequencing1. This technology provides sample barcoding that is compatible with any cell type or nucleus containing an accessible plasma membrane and allows for streamlined, pooled single-cell sample processing. The indices provide data quality advantages over non-indexed analysis methods in the form of doublet identification and retention of data from cells with low RNA content. MULTI-seq increases the efficiency and decreases the reagent cost of droplet-based single-cell RNA sequencing. Compatible with any droplet generation platform, the indexing system enables users to run and analyze up to 96 barcoded samples simultaneously in an 8-lane droplet platform. The MULTI-seq Lipid Modified Oligos kit is comprised of a lignoceric amide-modified anchor DNA oligo solution and a palmitic amide-modified co-anchor DNA oligo solution. Together, these lipid-modified oligos embed into cell or nuclei membranes and provide a landing pad for DNA barcodes with a complementary 5' sequence. The MULTI-seq Lipid Modified Oligos kit is intended for upstream sample preparation only, before pooling and single cell analysis. NOTE: Unique barcode oligos are not included and must be purchased separately. Barcode 3' oligo design with poly-A tail for mRNA enrichment1: Barcode oligo design for 5' cDNA library synthesis: 5'-CCTTGGCACCCGAGAATTCCA-8-base index-CCCATATAAGAAA-3' Custom barcode oligos may be ordered from SigmaAldrich.com/nextgenoligos. For minimal cross-contamination of barcode primers, order Next-Gen Sequencing Oligos with NGSO-Silver or NGSO-Gold quality. Reagents Provided • LMO001A Lignoceric Anchor with DNA Oligo • LMO001B Palmitic Co-anchor with DNA Oligo Each lipid-modified oligo is supplied at a concentration of 50 pM in water. Materials and reagents required but not provided • Trypsin (product number T2601, T2605, orT2610) • PBS (-), calcium and magnesium free PBS (product number D8537) • BSA (product number A4737, A3983, or similar) • Barcodes, 50 pM (custom ordered, NGSO-Silver or NGSO-Gold quality) This product is for R&D use only. Not for drug, household, or other uses. Please consult the Safety Data Sheet for information regarding hazards and safe handling practices. Store at -20 °C. At the supplied concentration, MULTI-seq Lipid Modified Oligos are stable for 1 year. Repeated freeze-thaws of MULTI-seq Lipid Modified Oligos are not recommended and may adversely affect functionality. Aliquot and freeze smaller volumes if multiple freeze-thaws are required. For research use only. Not for use in diagnostic procedures. 1

Preparation of Sample Tissue 1. Culture cells or obtain tissue (~500k or fewer cells per labeling condition). 2. Wash cells with PBS (-) twice. 3. Dissociate or lift cells to obtain single cell suspension (will vary depending on sample type). Note: Do not over-trypsinize cells for adherent cells. Over-trypsinized cells may break down during droplet generation after LMO labeling. 4. If cells are adherent, rinse and aspirate on plate. 5. If cells are already in suspension, centrifuge and rinse in tubes. 6. Ensure that cells are resuspended in buffer at single cell suspension prior to addition of...