NASC-seq2 Protocol

Using an automated pipetting platform or multichannel pipette, dispense 3uL of Vapor-Lock (Qiagen) to each well of a 384-well plate.

Note:

Do not use a non-contact dispenser (such as Dispendix IDOT or Formulatrix Mantis) for this step as the Vapor-Lock may severely damage the dispenser. We do this using an Agilent Bravo platform.

Prepare the following lysis buffer on ice.

A	B	C	D
Recombinant RNAse Inhibitor (40 U/uL)	2.5 u/uL	0.0188 uL	8.66 uL
4sU containing spUMI pool (OPTIONAL, 0.01 ng/uL)	0.04 pg/uL	0.0012 uL	0.553 uL
Triton-X100 (2 %)	0.1%	0.015 uL	6.91 uL
Nuclease-free water	-	0.265 uL	122.1 uL
Total		0.3 uL	138.2 uL

4On ice

Use a nanodispenser to distribute 0.3 uL of freshly prepared lysis buffer to each well of vapor-lock containing 384-well PCR plate.

0.3µL

On ice

Spin down at >3,000 G for 10 seconds.

3000x g

Optional: Store lysis plate.

If not immediately continuing with the next step, prepared lysis plates can be sealed with aluminium seals and stored at -80°C.

Grow cells in the presence of 50 μM 4sU. It can be helpful to collect untreated cells as a control.

Stop the labeling by transferring your cells to 15-ml falcon tubes on ice and wash them with cold PBS.

On ice

Distribute single cells into each well of the lysis plate by FACS sorting.

Spin down and seal the plates with aluminium seals and store them at -80°C.

Prepare the alkylation mix at room temperature.

A	B	C	D
Tris-HCL (pH 8.4, 1 M)	50 mM	0.03 uL	13.8 uL
DMSO (100 %)	45 %	0.24 uL	110.6 uL
IAA in DMSO (200 mM)	10 mM	0.03 uL	13.8 uL
Total		0.3 uL	138.2 uL

Please note that the calculations in this mix are calculated to the final volume in the alkylation reaction (i.e. 0.6uL). The DMSO final concentration listed above also accounts for the DMSO that is added with the IAA. Room temperature

Using a nanodispenser, distribute 0.3 uL of freshly prepared alkylation mix to all wells of the 384-well plate containing cells and lysis-buffer.

Spin the 384-well plate down at >3,000 x G for 10 seconds.

Incubate the plate at 50C for 15 minutes. While this is running, prepare the quenching mix (see the next section).

50°C

Prepare the quencing mix on ice.

A	B	C	D
DTT (1 M in H2O)	35 mM	0.035 uL	16.13 uL
dNTPs (10 mM each)	0.5 mM each	0.2 uL	92.16 uL
Oligo-dT (100 uM)	0.6 uM	0.024 uL	11.06 uL
Recombinant RNAse Inhibitor (40 U/uL)	0.4 u/uL	0.04 uL	18.43 uL
Nuclease-free water	-	0.101 uL	46.54 uL
Total		0.4 uL	184.32

Note that the dNTPs, oligo-dT and RRI concentrations above are calculated to the final volume in the Reverse Transcription mix (4uL) and not the Quenching mix (1uL).

Using a nanodispenser, distribute 0.4 uL of freshly prepared quenching and denuturation mix to all wells of the 384-well plate.

Spin the 384-well plate down at >3,000 x G for 10 seconds

Incubate the plate for 5 minutes at room temperature, followed by 10 minutes at 72C and a final hold at 4C. While this is running, prepare the Reverse Transcription mix (see the next section)

Prepare the Reverse Transcription mix as below

A	B	C	D
Tris-HCL (1 M, pH 8.0)	25 mM	0,1 uL	46.08 uL
NaCl (1 M)	35 mM	0,14 uL	64.51 uL
GTP (100 mM)	1 mM	0,04 uL	18.43 uL
MgCl2 (1 M)	2.5 mM	0.01 uL	4.61 uL
PEG (40 %)	5 %	0.5 uL	230.4 uL
DTT (1 M)	2 mM + carry-over from quenching and denaturation mix	0.008 uL	3.69 uL
Recombinant RNAse Inhibitor (40 U/uL)	0.4 U/uL + carry-over from lysis as well as quenching and denaturation mix	0.04 uL	18.43 uL
Template Switching Oligo (1 mM)	2 uM	0.008 uL	3.69 uL
Maxima H-minus RT enzyme (200 U/uL)	2 U/uL	0.04 uL	18.43 uL
H2O	-	2.11 uL	974.13 uL
Total		3 uL	1382.4 uL

Dispense 3 uL of the freshly prepared Reverse Transcription mix to all wells of the 384-well plate.

Spin the 384-well plate down at >3,000 x G for 10 seconds

Place the plate in the thermal cycler and start the Reverse Transcription program

For the remainer of the protocol we are using standard Smart-seq3 reaction conditions.

A detailed description of the Smart-seq3 protocol is also available on protocols.io.

Smart-seq3 Protocol

For convenience, the preamplification PCR protocol is also included below.

Prepare the preamplification PCR mix as below

A	B	C	D
Kapa HiFi HotStart buffer (5 X)	1 X	2.0 uL	820 uL
dNTPs (25 mM/each)	0.3 mM/each	0.12 uL	49.2 uL
MgCl2 (100 mM)	0.5 mM	0.05 uL	20.5 uL
Fwd Primer (100 uM)	0.5 uM	0.05 uL	20.5 uL
Rev Primer (100 uM)	0.1 uM	0.01 uL	4.1 uL
Polymerase (1 U/uL)	0.02 U/uL	0.2 uL	82 uL
Nuclease Free Water	-	3.57 uL	1463.7 uL
Total		6 uL	2460 uL

Preamplification PCR mix from Hagemann-Jensen et al.

Dispense 6 uL of the freshly prepared preamplification PCR mix to all wells of the 384-well plate.

Spin the 384-well plate down at >3,000 x G for 10 seconds

Place the plate in the thermal cycler and start the following PCR program

A	B	C	D
Step	Temperature	Time	Cycles
Initial denaturation	98 °C	3 min	1x
Denaturation	98 °C	20 sec	20-25x
Annealing	65°C	30 sec
Elongation	72 °C	4 min
Final Elongation	72 °C	5 min	1x
Hold	4 °C	Hold

For details on the purification of the amplified cDNA, the quality control and the tagmentation to produce the final sequencing libraries, please refer to the Smart-seq3 protocols.io.

Smart-seq3 Protocol

We have chosen to perform relatively long short-read sequencing with most data produced for NASC-seq2 being sequenced PE200. We highly recommend this as it will increase your ability to call individual molecules as either new (labeled) or old (unlabeled).

For details on data processing and analysis, please see the lab github:

https://github.com/sandberg-lab/NASC-seq2