snmCAT_V2

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.

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.
  Note

  In recent experiments, we found the RNA integrity from frozen human tissues may various. DO RIN analysis in bulk tissue before starting the experiments will be helpful to know the sample quality.

Prepare the stock solutions for nuclei isolation, stored at 4°C :

1. 10X Diluent buffer : Tris-Cl pH 8.0 (120 mM), KCl (150 mM), MgCl2(30 mM)
2. NIB: Tris-Cl pH 8.0 (10 mM), KCl (25 mM), MgCl2 (5 mM), Sucrose (250 mM)

Prepare the following solutions freshly before each experiment:

1. NIB_plus4On ice : NIB + DTT (1 mM) + Proteinase inhibitor (0.5X) + SUPERase• In ( 1:1000 dilution) + RNaseOUT ( 1:1000 dilution)
2. NIBTOn ice : NIB_plus + 0.1% Triton X-100
3. 50% IodixanolRoom temperature : 5 vol. Optiprep (60% Iodixanol) + 1 vol. Dilutent
4. 25% IodixanolRoom temperature : 1 vol. 50% Iodixanol + 1 vol. NIB
5. DPBS + RNase inhibitor On ice : DPBS + SUPERase• In ( 1:1000 dilution) + RNaseOUT ( 1:1000 dilution)

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 .

Transfer tissue sample or pre-ground tissue powder into the Dounce containing 3 ml of NIBT.1. Gently do douncing with a loose pestle (A) 40 times and then with a tight pestle (B) 40 times without introducing bubbles.

1. Mix the suspension with 2 ml of 50% Iodixanol by pipetting in 5ml ice-cold microcentrifuge tube.
2. Slowly pipette 1ml of cell mixture onto 500 µl 25% Iodixanol cushion, 5 tubes in total.
3. Centrifuge at 10,000 g for 20 min at 4°C using a swing rotor.
   Note

   Before adding cell mixture, we usually aliquot the 500 µl 25% Iodixanol cushion into 2 ml low retention microcentrifuge tubes and centrifuge at 10,000 g for 5 min to sharp the liquid interface.

Depending on specific experiment, proceed either Section A or B or C or A+B or C+A or C+A+B

Remove supernatant. Re-suspend the pellet in 1 ml of ice-cold DPBS + RNase Inhibitors.1. Add Hoechest 33342, then incubate on ice for 5 min.

Remove supernatant. Resuspend the pellet in 900 µl of DPBS + RNase inhibitors and 100 µl UltraPure BSA (50 mg/ml).1. 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)

Before do NOMe treatment, it's better to count the nuclei number either using hemocytometer or automated cell counter.1. 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.

1. Remove supernatant and resuspend in 50 µL DPBS.
2. Then make the 1st cycle NOMe reaction:

A	B
GpC Methyltransferase mix (per Rxn)	ul vol.
Nuclei mix	50
GpC Methyltransferase Buffer (10X)	15
S-adenosylmethionine (SAM 32mM)	0.15
GpC MTase	15
H2O	70

Incubate at 37°C for 8 mins

4. 2nd cycle NOMe reaction:

A	B
GpC Methyltransferase mix (per Rxn)	ul vol.
1st GpC treated nuclei mixture	150
GpC Methyltransferase Buffer (10X)	15
S-adenosylmethionine (SAM 32mM)	0.15
GpC MTase	15
H2O	120

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

Centrifuge at 1000 x g for 10 min at 4°C.1. Remove supernatant. Resuspend the pellet in 1ml DPBS + RNase inhibitors.

1. Filter with 40 um Cell strainer

Ready to run sorting.

Prepare mCT master mix:

A	B	C	D
Reagent	1 Rxn (μl)	384 Rxns (with 100% extra, μl)	8 x 384 Rxns (with 63% extra, μl)
Number of 384w plates		1	8
Rxn		800	5000
5X First-Strand Buffer	0.2	160	1000
0.1M DTT	0.05	40	250
1% Triton X-100	0.1	80	500
25mM MgCl2	0.1	80	500
500mM NaCl2	0.06	48	300
5-methyl-dNTP (10mM)	0.05	40	250
dT30VN_5 (100 μM)	0.012	9.6	60
N6_3 (100 μM)	0.02	16	100
TSO_4 (48 μM)	0.05	40	250
RNaseOUT40U/μl	0.025	20	125
SUPERaseIn 20U/μl	0.025	20	125
Superscript II RT*	0.05	40	200
H2O	0.258	206.4	1340
Total	1	800	5000

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

Add 1µL RT mix into each well of a 384 well plate.

Quick centrifugation the plates and keepOn ice.

Sort single nuclei using BD Influx or other sorters into 384 well plates on one-drop single mode.

Incubate with a thermocycler

A	B	C
Temperature	Time	Cycles
25°C	5 mins	1x
42°C	90 mins	1x
50°C	2 mins	10x
42°C	2 mins
85°C	5 mins	1x
4°C	∞

Prepare cDNA amplification mix:

A	B	C	D
Reagent	1 Rxn (μl)	384 Rxns (with 60% extra, μl)	8 x 384 Rxns (with 30% extra, μl)
Number of 384w plates		1	8
Rxns	1	600	4000
KAPA2G Buffer A (5X)	0.8	480	3200
ISPCR23_3 (100 μM)	0.024	14.4	96
KAPA2G Robust HotStart DNA Polymerase (5 U/μL)	0.016	9.6	64
H2O	2.16	1296	8640
Total	3	1800	12000

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

Incubate with a thermocycler

A	B	C	D
Step	Temperature	Time	Cycles
Initial denaturation	95 °C	3 min	1x
Denaturation	95 °C	15 sec	11-15x
Annealing	60°C	30 sec
Elongation	72 °C	2 min
Final Elongation	72 °C	5 min	1x
Hold	4 °C	Hold

Prepare uracil digestion mix:

A	B	C	D
Reagent	1 Rxn (μl)	384 Rxns (with 50% extra, μl)	8 x 384 Rxns (with 50% extra, μl)
UDG (G5010)	0.5	287.5	2300
EB buffer	0.5	287.5	2300
Total	1	575	4600

Add 1µL RT mix into each well of a 384 well plate and incubate at 37°C for 30 mins.

Add 25µL Zymo direct bisulfite conversion reagent into each well of a 384 well plate.

Incubate with a thermocycler

A	B
Temperature	Time
98 °C	8 min
64 °C	3.5 hrs
4 °C	hold

Proceed to the snmC-seq2 library preparation protocol.

https://www.protocols.io/view/methyl-c-sequencing-of-single-cell-nuclei-snmc-seq-pjvdkn6

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