TITLE: NASC-seq2 Protocol
AUTHORS: Gert-Jan Hendriks, Daniel Ramsköld, Anton Larsson, Juliane Mayr, Christoph Ziegenhain, Michael Hagemann-Jensen, Leonard Hartmanis, rickard.sandberg

[DESCRIPTION]
Insights into transcriptional bursting kinetics and regulation have emerged from real-time nascent RNA imaging and analyses of static RNA counts over cells. Here, we developed sensitive single-cell profiling of newly transcribed (or new) RNA in cells (NASC-seq2) that can easily be applied to tens of thousands of single cells to help shed new light on bursting dynamics and coordination.

[STEPS]
SECTION: Prepare lysis plates
2. Prepare the following lysis buffer on ice.



 

on ice 

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

0.3 µL 
on ice 


SECTION: Prepare lysis plates
5. 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.
SECTION: Cell culture and FACS sorting
6. Grow cells in the presence of 50 μM 4sU. It can be helpful to collect untreated cells as a control.
 

SECTION: Cell culture and FACS sorting
7. Stop the labeling by transferring your cells to 15-ml falcon tubes on ice and wash them with cold PBS.
on ice 

SECTION: Cell culture and FACS sorting
8. Distribute single cells into each well of the lysis plate by FACS sorting.
SECTION: Cell culture and FACS sorting
9. Spin down and seal the plates with aluminium seals and store them at -80°C.
SECTION: Alkylation
10. Prepare the alkylation mix at room temperature.


 
Room temperature 


 

 

SECTION: Alkylation
11. 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.
SECTION: Alkylation
12. Spin the 384-well plate down at >3,000 x G for 10 seconds.
SECTION: Alkylation
13. Incubate the plate at 50C for 15 minutes. While this is running, prepare the quenching mix (see the next section).

50 °C 

SECTION: Quenching and Denaturation
14. Prepare the quencing mix on ice.


 

SECTION: Prepare lysis plates
4. Spin down at >3,000 G for 10 seconds.

3000 x g, 10 s 

SECTION: Quenching and Denaturation
15. Using a nanodispenser, distribute 0.4 uL of freshly prepared quenching and denuturation mix to all wells of the 384-well plate.
SECTION: Quenching and Denaturation
16. Spin the 384-well plate down at >3,000 x G for 10 seconds
SECTION: Quenching and Denaturation
17. 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)
SECTION: Reverse Transcription
18. Prepare the Reverse Transcription mix as below

 

SECTION: Reverse Transcription
19. Dispense 3 uL of the freshly prepared Reverse Transcription mix to all wells of the 384-well plate.
SECTION: Reverse Transcription
20. Spin the 384-well plate down at >3,000 x G for 10 seconds
SECTION: Reverse Transcription
21. Place the plate in the thermal cycler and start the Reverse Transcription program
SECTION: Preamplification PCR
22. 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.
 

For convenience, the preamplification PCR protocol is also included below.
SECTION: Preamplification PCR
23. Prepare the preamplification PCR mix as below


 

SECTION: Preamplification PCR
24. Dispense 6 uL of the freshly prepared preamplification PCR mix to all wells of the 384-well plate.
SECTION: Preamplification PCR
25. Spin the 384-well plate down at >3,000 x G for 10 seconds
SECTION: Preamplification PCR
26. Place the plate in the thermal cycler and start the following PCR program

 



SECTION: Purification, QC and Tagmentation
27. 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.

 

SECTION: Sequencing and data analysis
28. 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

SECTION: Prepare lysis plates
1. 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.