SUPPLEMENTAL USER GUIDE Chromium Next GEM Automated Single Cell 3ʹ cDNA Kit v3.1 FOR USE WITH Chromium Next GEM Automated Single Cell 3’ cDNA Kit v3.1, 24 rxns PN-1000424 PAIRS WITH DOCUMENTS Chromium Next GEM Automated Single Cell 3’ Reagent Kits v3.1 User Guide (CG000286) Chromium Connect Instrument User Guide (CG000180) Automated Library Construction User Guide (CG000474)

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Document Revision Summary Document Number Chromium Next GEM Automated Single Cell 3' cDNA Kit v3.1 Supplemental User Guide Revision Date Chromium Next GEM Automated Single Cell 3ʹ cDNA Kit v3.1 Supplemental User Guide • Rev A

Automated Modular Workflow Chromium Next GEM Automated Single Cell 3' cDNA Kit v3.1 Workflow Overview Chromium Next GEM Automated Single Cell 3ʹ cDNA Kit v3.1 Protocol Steps & Timing Chromium Next GEM Automated Single Cell 3ʹ cDNA Kit v3.1 Supplemental User Guide • Rev A

Automated Modular Workflow Chromium Next GEM Next GEM Automated Automated Single Single cDNA Kit v3.1 Cell 3' Cell 3' cDNA Kit v3.1 Workflow Overview Gene Expression This is a supplemental document that provides an overview of the Chromium Next GEM Automated Single Cell 3' Gene Expression modular workflow along with a list of 10x Genomics reagents needed to generate cDNA from single cell suspension. As part of this automated flexible workflow, cDNA can be generated using the Chromium Next GEM Automated Single Cell 3' cDNA Kit v3.1 (PN-1000424). The cDNA can be stored at 4°C or -20°C or used directly...

Automated Modular Workflow Chromium Next GEM Automated Single Cell 3ʹ cDNA Kit v3.1 Chromium Next GEM Automated Single Cell 3' cDNA Kit v3.1, 24 rxns PN-1000424 Reagent volumes and colors are different in each of the module types, not all module tubes contain reagents. Chromium Next GEM Automated Single Cell Kit, cDNA Module 1, 24 rxns PN-1000395 (store at 4°C) Chromium Next GEM Automated Single Cell Kit, cDNA Module 1, 24 rxns Single Cell cDNA Module 1 DynabeadsTM MyOneTM SILANE Chromium Next GEM Automated Single Cell Kit, cDNA Module 2, 24 rxns PN-1000396 (store at -20°C) Chromium Next GEM...

Automated Modular Workflow Chromium Next GEM Automated Single Cell 3' cDNA Kit v3.1, 24 rxns PN-1000424 Chromium Next GEM Automated Single Cell 3ʹ Gel Bead Kit v3.1, 24 rxns PN-1000137 (store at -80°C) Chromium Next GEM Automated Single Cell 3’ Gel Bead Kit v3.1, 24 rxns Single Cell 3’ v3.1 Gel Beads Use the 10x Genomics reagents listed above for automated cDNA generation. After cDNA generation, store cDNA until ready to proceed to Gene Expression Library Construction using the Automated Library Construction Kit (PN-1000428). Refer to Automated Library Construction User Guide (CG000474) for details....

Automated Modular Workflow   Protocol Steps & Timing The table below provides an overview of the complete automated modular workflow steps and timing Steps Manual Cell Preparation (Dependent on Cell Type) Gather & Load Reagents and Consumables Automated Single Cell Gene Expression cDNA Master Mix Preparation Chromium Automated Controller Loading GEM Generation OPTIONAL Confirm GEM Generation (Manual, 5 min) Post GEM RT-Cleanup – Dynabead cDNA Amplification cDNA Cleanup – SPRIselect Stop after SPRI clean up, store cDNA at 4°C for �72 hrs or -20°C for �4 wks cDNA QC & Quantification (Manual, 50...