Table 2:

The incubating procedure used in the protocol.

Incubating Procedure	Temperature(1)	Time
GEM-RT Incubation	Lid Temperature 53 °C
Step 1	53 °C	45 min
Step 2	85 °C	5 min
Step 3	4 °C	Hold
10x Genomics cDNA Amplification	Lid Temperature 105 °C
Step 1	98 °C	3 min
Step 2	98 °C	15 s
Step 3	63 °C	20 s
Step 4	72 °C	1 min
Step 5	Repeat steps 2 to 4 for 12 cycles in total (2)
Step 6	72 °C	1 min
Step 7	4 °C	Hold
Library construction	Lid Temperature 65 °C
Pre-cool block	4 °C	Hold
Fragmentation	32 °C	5 min
End Repair and A-tailing	65 °C	30 min
Hold	4 °C	Hold
Adaptor ligation	Lid Temperature 30 °C
Step 1	20 °C	15 min
Step 2	4 °C	Hold
Sample index PCR	Lid Temperature 105 °C
Step 1	98 °C	45 s
Step 2	98 °C	20 s
Step 3	54 °C	30 s
Step 4	72 °C	20 s
Step 5	Repeat steps 2 to 4 for 12 cycles in total (3)
Step 6	72 °C	1 min
Step 7	4 °C	Hold
Lipid barcode library PCR
Step 1	95 °C	5 min
Step 2	98 °C	15 s
Step 3	60 °C	30 s
Step 4	72 °C	30 s
Step 5	Repeat steps 2 to 4 for 10 cycles in total (4)
Step 6	72 °C	1 min
Step 7	4 °C	Hold
Antibody barcode library PCR
Step 1	95 °C	3 min
Step 2	95 °C	20 s
Step 3	60 °C	30 s
Step 4	72 °C	20 s
Step 5	Repeat steps 2 to 4 for 8 cycles in total (5)
Step 6	72 °C	5 min
Step 7	4 °C	Hold

⁽¹⁾Pay attention to the different lid temperature used in every Procedure.

⁽²⁾Set total cycle numbers according to the cell load: 13 cycles for <500 cell load; 12 cycles for 500–6,000 cell load; 11 cycles for >6,000 cell load.

⁽³⁾Set total cycle numbers according to the cDNA input: 14–16 cycles for 1–25 ng cDNA; 12–14 cycles for 25–150 ng cDNA; 10–12 cycles for 150–500 ng cDNA; 8–10 cycles for 500–1,000 ng cDNA; 6–8 cycles for 1000–1500 ng cDNA.

⁽⁴⁾Set total cycle numbers according to the cDNA input: 8–12 cycles.

⁽⁵⁾Set total cycle numbers according to the cDNA input: 6–10 cycles.