TITLE: snmCAT_V1
AUTHORS: Bang-An Wang,  Chongyuan Luo, Joseph Ecker

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">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.</div></div>

[STEPS]
?. [Background]
This protocol is based on the originial 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.
?. [Nuclei preparation]
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.
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.
?. [Nuclei preparation]
Prepare the stock solutions for nuclei isolation, stored at  :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)
4 °C
?. [Nuclei preparation]
Prepare the following solutions freshly before each experiment:NIB_plus : NIB + DTT (1 mM) + Proteinase inhibitor (0.5X) + SUPERase• In ( 1:1000 dilution) + RNaseOUT ( 1:1000 dilution)NIBT : NIB_plus + 0.1% Triton X-10050% Iodixanol : 5 vol. Optiprep (60% Iodixanol) + 1 vol. Dilutent25% Iodixanol : 1 vol. 50% Iodixanol + 1 vol. NIBDPBS + RNase inhibitor  : DPBS + SUPERase• In ( 1:1000 dilution) + RNaseOUT ( 1:1000 dilution)
on ice
on ice
0 Room temperature
0 Room temperature
on ice
?. [Nuclei preparation]
Pre-chilling steps:Pre-chill 2 ml and 5 ml low retention microcentrifuge tubeCooling down the swing bucket rotor for centrifuging .
on ice
4 °C
?. [Nuclei preparation]
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 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.
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.
?. [Nuclei preparation]
Depending on specific experiment, proceed either Section A or B or C or A+B or C+A or C+A+B
?. [Section_A_Nuclei staining_ONLY ]
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_B_Ab staining]
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_C_NOMe treatment]
Remove supernatant. Resuspend each pellet in 200 µl of ice-cold NIB.Count the number of nuclei. Divide the nuclei suspension to vials each containing 1M nuclei.Centrifuge at 2000 x g for 10 min at 4°C.Remove supernatant. Resuspend the pellet in 200 µl of GpC methyltransferase + RNase Inhibitors reaction.Incubate at 37°C for 10 min.Add 800 µl of ice-cold DPBS + RNase Inhibitors to the GpC methyltransferase reaction.GpC Methyltransferase mix (per Rxn)ul vol.1M Sucrose60GpC Methyltransferase Buffer (10X) 20S-adenosylmethionine (SAM 32mM)2GpC MTase20H2O98
GpC Methyltransferase mix (per Rxn)ul vol.1M Sucrose60GpC Methyltransferase Buffer (10X) 20S-adenosylmethionine (SAM 32mM)2GpC MTase20H2O98
?. [Pre_sorting]
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 strainerReady to run sorting
?. [Prepare collection plates]
Prepare mCT master mix:Reagent1 Rxn (μl)384 Rxns (with 100% extra, μl)8 x 384 Rxns (with 100% extra, μl)Number of 384w plates185X First-Strand Buffer0.2153.61228.80.1M DTT0.0538.4307.21% Triton X-1000.176.8614.450mM MgCl20.0538.4307.25-methyl-dNTP (10mM)0.0538.4307.2dT30VN_4 (100 μM)0.017.6861.44N6_2 (100 μM)0.0215.36122.88TSO_3 (100 μM)0.0215.36122.88RNaseOUT 40U/μl0.017.6861.44SUPERaseIn 20U/μl0.017.6861.44Superscript II RT0.017.6861.44H2O0.47360.962887.68Total17686144Use Beckman i7 robot to distribute mct reaction buffer to 384-plates:Add  RT mix into each well of a 384 well plate.Quick centrifugation the plates and keep .
Reagent1 Rxn (μl)384 Rxns (with 100% extra, μl)8 x 384 Rxns (with 100% extra, μl)Number of 384w plates185X First-Strand Buffer0.2153.61228.80.1M DTT0.0538.4307.21% Triton X-1000.176.8614.450mM MgCl20.0538.4307.25-methyl-dNTP (10mM)0.0538.4307.2dT30VN_4 (100 μM)0.017.6861.44N6_2 (100 μM)0.0215.36122.88TSO_3 (100 μM)0.0215.36122.88RNaseOUT 40U/μl0.017.6861.44SUPERaseIn 20U/μl0.017.6861.44Superscript II RT0.017.6861.44H2O0.47360.962887.68Total17686144
1 µl
on ice
?. [FACS]
Sort single nuclei using BD Influx or other sorters into 384 well plates on one-drop single mode.
?. [Reverse Transcription]
Incubate with a thermocycler25°C5 mins42°C90 mins70°C15 mins4°C∞
25°C5 mins42°C90 mins70°C15 mins4°C∞
?. [PCR Amplification]
Prepare cDNA amplification mix: Reagent1 Rxn (μl)384 Rxns (with 30% extra, μl)8 x 384 Rxns (with 30% extra, μl)Number of 384w plates18mCT-seq RT reaction15004000KAPA2G Buffer A (5X)0.84003200ISPCR23_2 (100 μM)0.0241296KAPA2G Robust HotStart DNA Polymerase (5 U/μL)0.016864H2O2.1610808640Total4200016000Add  RT mix into each well of a 384 well plate.Incubate with a thermocyclerStepTemperatureTimeCyclesInitial denaturation95 °C3 min1xDenaturation95 °C15 sec11-15xAnnealing60°C30 secElongation72 °C2 minFinal Elongation72 °C5 min1xHold4 °CHold
Reagent1 Rxn (μl)384 Rxns (with 30% extra, μl)8 x 384 Rxns (with 30% extra, μl)Number of 384w plates18mCT-seq RT reaction15004000KAPA2G Buffer A (5X)0.84003200ISPCR23_2 (100 μM)0.0241296KAPA2G Robust HotStart DNA Polymerase (5 U/μL)0.016864H2O2.1610808640Total4200016000
3 µl
StepTemperatureTimeCyclesInitial denaturation95 °C3 min1xDenaturation95 °C15 sec11-15xAnnealing60°C30 secElongation72 °C2 minFinal Elongation72 °C5 min1xHold4 °CHold
*Each cell type contains a different RNA quantity per cell or nucleus. The optimal cycle number for cDNA amplification needs to be optimized for each cell type. Mouse and human neuronal nuclei - 14 cyclesMouse and human non-neuronal nuclei - 16 cyclesHuman H1 and HEK293 whole cell - 12 cyclesHuman H1 and HEK293 nuclei - 15 cycles
?. [UDG Diegestion]
Prepare uracil digestion mix:Reagent1 Rxn (μl)384 Rxns (with 50% extra, μl)8 x 384 Rxns (with 50% extra, μl)UDG (G5010)0.5287.52300EB buffer0.5287.52300Total15754600Add  RT mix into each well of a 384 well plate and incubate at 37°C for 30 mins.
Reagent1 Rxn (μl)384 Rxns (with 50% extra, μl)8 x 384 Rxns (with 50% extra, μl)UDG (G5010)0.5287.52300EB buffer0.5287.52300Total15754600
1 µl
?. [Bisulfite conversion]
Add  Zymo direct bisulfite conversion reagent into each well of a 384 well plate.Incubate with a thermocyclerTemperatureTime98 °C8 min64 °C3.5 hrs4 °Chold
25 µl
TemperatureTime98 °C8 min64 °C3.5 hrs4 °Chold
?. [snmC-Seq2 library preparation]
Proceed to the snmC-seq2 library preparation protocol.https://www.protocols.io/view/methyl-c-sequencing-of-single-cell-nuclei-snmc-seq-pjvdkn6