TITLE: snmCAT_V2
AUTHORS: Bang-An  Wang,    Chongyuan Luo, Hanqing  Liu, Anna  Bartlett, Rosa  Castanon, Joseph Ecker

[DESCRIPTION]
To comprehensively assess the molecular phenotypes of single cells in tissues, we devised single-nucleus methylCytosine, Chromatin accessibility and Transcriptome sequencing (snmCAT-seq) and applied it to various sample sources, like culture cells, fresh/frozen mice tissues (brain, liver, pancreases etc) and postmortem human frontal cortex tissue.

[GUIDELINES]
For details, please refer to the publications below:

Luo, C., Rivkin, A., Zhou, J., Sandoval, J.P., Kurihara, L., Lucero, J., Castanon, R., Nery, J.R., Pinto-Duarte, A., Bui, B., et al. (2018). Robust single-cell DNA methylome profiling with snmC-seq2. Nat. Commun. 9, 3824.

Luo, C., Liu, H., Xie, F., Armand, E.J., Siletti, K., Bakken, T., Fang, R., Doyle, W.I., Hodge, R.D., Hu, L., et al. (2019). Single nucleus multi-omics links human cortical cell regulatory genome diversity to disease risk variants. bioRxiv.

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[STEPS]
SECTION: Background
1. 
This protocol is based on the original protocol named as snmC2T-seq from the BioRxiv paper (ChongyuanLuo, et al. BioRxiv 2019)  https://www.biorxiv.org/content/10.1101/2019.12.11.873398v1 and SMART-seq3 (Hagemann-Jensen, et al.Nat Biotechnol 2020, https://www.protocols.io/view/smart-seq3-protocol-bcq4ivyw. 

Reagents and oligo sequence can be found in Materials part.

SECTION: Nuclei preparation
2. Sample preparation:

We typically grind tissue samples with liquid nitrogen ahead of time and stored at -80°C.
For smaller mouse tissues, we usually snap freeze the fresh dissected samples and store at -80°C.
For culture cells, we typically pellet either suspension cells or dissociated adherent cells, aspirate supernatant then store at -80°C.
 

SECTION: Nuclei preparation
2.1. Prepare the stock solutions for nuclei isolation, stored at 4 °C :

10X Diluent buffer : Tris-Cl pH 8.0 (120 mM), KCl (150 mM), MgCl2(30 mM)
NIB: Tris-Cl pH 8.0 (10 mM), KCl (25 mM), MgCl2 (5 mM), Sucrose (250 mM)
SECTION: Nuclei preparation
2.2. Prepare the following solutions freshly before each experiment:

NIB_pluson ice : NIB + DTT (1 mM) + Proteinase inhibitor (0.5X) + SUPERase• In ( 1:1000 dilution) + RNaseOUT ( 1:1000 dilution)
NIBTon ice : NIB_plus + 0.1% Triton X-100
50% IodixanolRoom temperature : 5 vol. Optiprep (60% Iodixanol) + 1 vol. Dilutent
25% IodixanolRoom temperature : 1 vol. 50% Iodixanol + 1 vol. NIB
DPBS + RNase inhibitor on ice : DPBS + SUPERase• In ( 1:1000 dilution) + RNaseOUT ( 1:1000 dilution)
SECTION: Nuclei preparation
2.3. Pre-chilling steps:
Plunge the Dounce and Pestles on ice (in a 50ml tube to avoid contamination from ice).  Transfer 3ml of NIBT buffer to the Dounce in ice and let them chill for 10 min.
Pre-chill 2 ml and 5 ml low retention microcentrifuge tubeon ice 
Cooling down the swing bucket rotor for centrifuging4 °C .
SECTION: Nuclei preparation
3. Transfer tissue sample or pre-ground tissue powder into the Dounce containing 3 ml of NIBT.
Gently do douncing with a loose pestle (A) 40 times and then with a tight pestle (B) 40 times without introducing bubbles.
Mix the suspension with 2 ml of 50% Iodixanol by pipetting in 5ml ice-cold microcentrifuge tube.
Slowly pipette 1ml of cell mixture onto 500 µl 25% Iodixanol cushion, 5 tubes in total.
Centrifuge at 10,000 g for 20 min at 4°C using a swing rotor.
 



SECTION: Nuclei preparation
4. Depending on specific experiment, proceed either Section A or B or C or A+B or C+A or C+A+B
SECTION: Section_A_Nuclei staining_ONLY
5. Remove supernatant. Re-suspend the pellet in 1 ml of ice-cold DPBS + RNase Inhibitors.
Add Hoechest 33342, then incubate on ice for 5 min.

SECTION: Section_B_Ab staining
6. Remove supernatant. Resuspend the pellet in 900 µl of DPBS + RNase inhibitors and 100 µl UltraPure BSA (50 mg/ml).
Add specific amount of nucleus antibodies and incubate on ice for 20 min. 
(For mouse/human neurons, 1 µl AlexaFluor 488 conjugated anti-NeuN clone A60 is used)
SECTION: Section_C_NOMe treatment
7. Before do NOMe treatment, it's better to count the nuclei number either using hemocytometer or automated cell counter.
Transfer less than 1 million nuclei per reaction to a new tube, centrifuge at 1000 x g for 10 min at 4°C to spin down the nuclei.
Remove supernatant and resuspend in 50 µL DPBS. 
Then make the 1st cycle NOMe reaction:

 

 Incubate at 37°C for 8 mins

4. 2nd cycle NOMe reaction:

 
 Incubate at 37°C for 8 mins

5. Stop the reaction in 1ml ice-cold DPBS and centrifuge at 1000 x g for 10min at 4°C
SECTION: Pre_sorting
8. Centrifuge at 1000 x g for 10 min at  4°C.
Remove supernatant. Resuspend the pellet in 1ml DPBS + RNase inhibitors.
Filter with 40 um Cell strainer

Ready to run sorting.
SECTION: Prepare collection plates
9. 
Prepare mCT master mix:

 

Use Beckman i7 robot to distribute mct reaction buffer to 384-plates:

Add 1 µL RT mix into each well of a 384 well plate.
Quick centrifugation the plates and keepon ice.

 

SECTION: FACS
10. Sort single nuclei using BD Influx or other sorters into 384 well plates on one-drop single mode.
SECTION: Reverse Transcription
11. Incubate with a thermocycler

 


SECTION: PCR Amplification
12. Prepare cDNA amplification mix: 

 
Add 3 µL RT mix into each well of a 384 well plate.

Incubate with a thermocycler

 

 


SECTION: UDG Diegestion
13. Prepare uracil digestion mix:

 
Add 1 µL RT mix into each well of a 384 well plate and incubate at 37°C for 30 mins.
SECTION: Bisulfite conversion
14. Add 25 µL Zymo direct bisulfite conversion reagent into each well of a 384 well plate.
Incubate with a thermocycler


 



SECTION: snmC-Seq2 library preparation
15. Proceed to the snmC-seq2 library preparation protocol.
https://www.protocols.io/view/methyl-c-sequencing-of-single-cell-nuclei-snmc-seq-pjvdkn6
SECTION: snmC-Seq2 library preparation
16. The NGS mapping pipeline and analysis tools can be found in the packages coded by Hanqing:
Yap: https://hq-1.gitbook.io/mc/
Allcools: https://lhqing.github.io/ALLCools/intro.html