SMART-Seq

Dilute the oligo-dT30VN primer to 10micromolar (µM) by adding 10µL of 100micromolar (µM) oligo-dT primers and 90µL of nuclease-free water to a tube and mix well.

Prepare cell lysis buffer by adding 1µL of RNase inhibitor to 19µL of a 0.2% (vol/vol) Triton X-100 solution.

Isolate single cells in the lowest possible volume (preferably ≤0.5µL, possibly 0.3µL) or pipet the appropriate amount of RNA into a 0.2mL thin-walled PCR tube. Single cells can be obtained either by using a micro capillary pipette or via FACS.

Place each single cell into a 0.2mL thin-walled PCR tube containing 2µL of cell lysis buffer, 1µL of oligo-dT primer and 1µL of dNTP mix.

Quickly vortex the tube to mix, and then spin down the solution (700g for 0h 0m 10s at Room temperature) and immediately place it On ice.

Incubate the samples at 72°C for 0h 3m 0s and immediately put the tube back 72On ice.

Spin down the samples (700g for 0h 0m 10s at 72Room temperature) to collect the liquid at the bottom of the tubes, and then put them immediately back 72On ice.

Purified RNA:

Xul RNA up to 2.5ul

1ul oligo-dT Primer (10uM)

1ul dNTP (10mM)

xul H2O

4.5ul Total

72°C for 0h 3m 0s, snap cool.

Prepare the RT mix for all reactions plus one additional reaction by combining and mixing the reagents listed in the table below.

A	B	C
Component	Volume (ul)	Final Conc
Superscipt II	0.50	100 U
RNAse inhibitor (40 U/ul)	0.25	10 U
Superscript II FS buffer (5X)	2.00	1X
DTT (100 mM)	0.50	5 mM
Betaine (5 M)	2.00	1 M
MgCl2 (1 M)	0.06	6 mM
TSO (100uM)	0.10	1 uM
H2O	0.29	-
Total	5.70	-

Add 5.7µL of RT mix to Samples for a total of 10µL.

Spin and incubate as follows:

A	B	C	D
Cycle	Temperature (°C)	Time	Purpose
1	42	90 min	RT and template-switching
2–11	50	2 min	Unfolding of RNA secondary structures
	42	2 min	Completion/continuation of RT and template-switching
12	70	15 min	Enzyme inactivation
13	4	Hold	Safe storage

Prepare the PCR mix for all reactions plus one additional reaction by combining and mixing the following components:

A	B	C
Component	Volume (μl)	Final concentration
First-strand reaction	10	–
KAPA HiFi HotStart ReadyMix (2×)	12.50	1×
IS PCR primers (10 μM)	0.25	0.1 μM
Nuclease-free water	2.25	–
Total volume	25	–

Add 15µL of PCR mix to each tube from Step 12, which contains the first-strand reaction and perform the PCR in a thermal cycler by using the following program:

A	B	C	D	E
Cycle	Denature	Anneal	Extend	Hold
1	98 °C, 3 min	–	–	–
2–19 (see below)	98 °C, 20 s	67 °C, 15 s	72 °C, 6 min	–
20	–	–	72 °C, 5 min	–
21	–	–	–	4 °C

A	B	C
Input Amount Total RNA	Input Amount, Cells	Typical No. of PCR Cycles
10 ng	1,000 cells	12
1 ng	100 cells	12
500 pg	50 cells	13
100 pg	10 cells	15
10 pg	1 cell	18

Perform a typical Ampure cleanup using 1:1 ratio of Ampure:cDNA.

Elute using 17.5µL EB solution and pipette 15µL to transfer to a new tube.

Run a High Sensitivity Bioanalyzer Chip to check for quality of cDNA.

A good library should be free of short (<500 bp) fragments and should show a peak at 1.5–2 kb.

Setup the tagmentation RXN as follows:

A	B	C
Component	Volume (μl)	Final concentration
Tagment DNA buffer (TD, 2×)	10	1×
Amplicon tagment mix	5	–
DNA from PCR	Variable	–
Nuclease-free water	Variable	–
Total volume	20	–

Incubate in a thermal cycler at 55°C for 0h 5m 0s and bring to 4°C HOLD.

Add 5µL of NT buffer to the 20µL RXN and mix.

Incubate at 4Room temperature for 0h 5m 0s.

Prepare the following PCR RXN as follows:

A	B
Component	Volume (μl)
DNA	25
Nextera PCR master mix	15
Index 1 primers (N7xx)	5
Index 2 primers (N5xx)	5
Total volume	50

Run the PCR RXN on a thermal cycler with the following conditions:

A	B	C	D	E
Cycle	Denature	Anneal	Extend	Hold
1	–	–	72 °C, 3 min	–
2	95 °C, 30 s	–	–	–
3–14*	95 °C, 10 s	55 °C, 30 s	72 °C, 30 s	–
15	–	–	72 °C, 5 min	–
16	–	–	–	4 °C

*for 1ng, 8-12 cycles could be used