Protocol Steps & Timing

Steps	Timing
Nuclei Isolation
Dependent on Cell Type	~1-2 h
Choose either Individual (Step 1) or Scalable (Step 2) Transposition
Step 1: Individual Transposition
1.1 Prepare Transposition Mix	10 min
1.2 Isothermal Incubation	30-60 min
1.3 Post Transposition Wash	10 min
OR
Step 2: Scalable Transposition
2.1 Prepare Transposition Mix	10 min
2.2 Isothermal Incubation	30-60 min
2.3 Post Transposition Wash	10 min
Step 3: GEM Generation & Barcoding
3.1 Prepare Master Mix	10 min
Contents	10 min
3.3 Run the Chromium X Series Instrument (X/iX)	6 min
3.4 Transfer GEMs	3 min
3.5 GEM Incubation	75 min
3.6 Quenching Reaction	5 min	−80°C ≤ 4 week
Step 4: Post GEM Incubation Cleanup
4.1 Post GEM Incubation Cleanup – Dynabeads	35 min
4.2 Post GEM Incubation Cleanup – SPRIselect	15 min
Step 5: Pre Amplification PCR
5.1 Prepare Pre Amplification Mix	10 min
5.2 Pre Amplification PCR	30 min	4°C ≤18 h
5.3 Pre Amplification SPRI Cleanup	15 min	4°C ≤72 h or −20°C long-term
Step 6: ATAC Library Construction
6.1 Sample Index PCR	45 min
6.2 Post Sample Index Double Sided Size Selection – SPRIselect	20 min	4°C ≤72 h or −20°C long-term
6.3 Post Library Construction QC	60 min
Step 7: cDNA Amplification
7.1 cDNA Amplification	40 min	4°C ≤72 h or −20°C ≤1 week
7.2 cDNA Cleanup – SPRIselect	15 min	4°C ≤72 h or −20°C ≤4 weeks
7.3 cDNA QC & Quantification	50 min
Step 8: Gene Expression Library Construction
8.1 Fragmentation, End Repair & A-tailing	45 min
8.2 Post Fragmentation, End Repair & A-tailing Double Sided Size Selection – SPRIselect	30 min
8.3 Adaptor Ligation	25 min
8.4 Post Ligation Cleanup – SPRIselect	20 min
8.5 Sample Index PCR	40 min	4°C ≤72 h
8.6 Post Sample Index PCR Double Sided Size Selection – SPRIselect	30 min	4°C ≤72 h or -20°C long term
8.7 Post Library Construction QC	50 min