TITLE: KAPP-Sen TMC: Nuclei Suspension Preparation for snPATHO-seq
AUTHORS: Dylan Baker, Juliana Alcoforado Diniz, Jessica Garofalo, Paul Robson

[DESCRIPTION]
This protocol for nuclei suspension preparation was adapted from Vallejo et al. (https://doi.org/10.1101/2022.08.23.505054) with minor changes. Once the suspension was prepared we proceeded to the Chromium X run using Chromium Fix RNA Profiling by 10x Genomics.

[STEPS]
SECTION: Reagents and Consumables
1. Hyaluronidase: 0.5mg/ml
Liberase TM: 1mg/ml
Collagenase D:  1mg/ml
SECTION: Nuclei isolation
1.1. Cut up to 2 ~25 μm-thick sections and place it in 1.5 mL Eppendorf tube. Store dry at 4°C if not used immediately. To keep it dry, you may use the cylinder containing silica beads that comes with 10x Genomics chips. 
SECTION: Nuclei isolation
1.2. Wash sections three times with 1 mL Xylene (add to the tube with curls) for 10’ to remove the paraffin, rehydrate in sequential 1’ of 1 mL ethanol immersions (2× 100%, followed by 1× 70%, 50% and 30% ethanol). IMPORTANT: make sure paraffin is fully removed or digestion will be suboptimal. 
SECTION: Nuclei isolation
1.3. Wash 3 times for 1' (2x1 ml wash and 1x800 ul final wash) with 1x PBS + 0.5 mM CaCl2
SECTION: Nuclei isolation
1.4. Remove as much volume as possible, and digest tissue for 45-60’(*) at 37°C(**) in 1 mL of RPMI1640 supplemented with 0.25-1 mg/ml Liberase TM(***) + 0.25-1 mg/ml Collagenase D(***) + 0.25-1 mg/ml Hyaluronidase + 1 U/ul RNAse Inhibitor.(*) NOTE: some blocks require longer digestion time, so inspect visually and help dissociation by pipetting up and down with a P1000 pipette (pipette up/down every 10-15 mins). (**) Incubation is done in a Thermomixer 800 rpm. IMPORTANT: dissociation does not need to be complete; the objective here is to loosen up the material to facilitate the nuclei release. Dissociation completeness varies from block to block. Tissue does not need to be fully digested.
SECTION: Nuclei isolation
1.5. After digestion, add 400 uL of Ez Lysis Buffer to the sample, mix by inverting 5× and centrifuge for 5’ at 850xRCF at 4°C.
SECTION: Nuclei isolation
1.6. Resuspend the pellets (released nucs and undigested tissue) in 250 uL Ez Lysis buffer + 2% BSA + 1 U/uL RNAse Inhibitor and homogenize the sample using a douncer/pestle by stroking 10-20 times.
SECTION: Nuclei isolation
1.7. After homogenization add 750 uL Ez Lysis buffer + 2% BSA + 1 U/uL RNAse Inhibitor and continue disaggregating by pipetting using a P1000 pipette (10 times). Incubate on ice for 15’. At 5’ mark pipette up and down using a P1000 pipette (10 times).
SECTION: Nuclei isolation
1.8. Pass sample through a 70 μm PluriStrainer filter (not Flowmi!) and centrifuge the flowthrough for 5’ at 850xRCF at 4°C. This is to remove big chunky, indigested tissue. 
SECTION: Nuclei isolation
1.9. Wash nuclei suspensions once more with 800 uL of EzLysis buffer + 2% BSA + 1 U/uL RNAse Inhibitor (very gentle).
SECTION: Nuclei isolation
1.10. Pellet the nuclei for 5’ at 850xRCF at 4°C and wash nuclei twice PBS 0.5x + 0.02% BSA and resuspend in 300 uL of PBS 0.5x + 0.02% BSA (resuspension volume can vary depending on pellet size). Pass sample through a 50 μm PluriStrainer filter (not Flowmi!).
SECTION: Nuclei isolation
1.11. Count using Luna-FX7 or similar based on dual-fluorescence such asAO/PI.   
SECTION: Storage
2. If not proceeding to the Chromium X run using Chromium Fix RNA Profiling (10x Genomics), the user could supplement the sample with 0.1x volume of Enhancer solution (10x Genomics) + 10% Glycerol, rest on ice for 10’ and cryopreserve at −80°C.