TITLE: Intranuclear CITE-seq (inCITE-seq): joint single-cell measurements of multiplexed nuclear proteins and RNA 
AUTHORS: Hattie  Chung, Emma  Magee

[DESCRIPTION]
This protocol allows for intranuclear antibody staining of fixed nuclei in suspension. Nuclei suspensions are suitable for CITE-seq and 10X Genomic applications.

[STEPS]
SECTION: Fixation and Permeabilization
21. Transfer 1 mL of nuclei suspension from the filter tube into a pre-chilled 15mL Falcon tube. 

SECTION: Primary Antibody Stain
29. Resuspend pellet in 500 µL of Blocking Buffer. Pipette up and down multiple times using a P200 pipette to ensure the sample is mixed thoroughly. 

SECTION: Fixation and Permeabilization
24. Incubate for 10 min  at 4 °C while rocking.

SECTION: Nuclei Extraction
4. Remove frozen tissue sample from -80 °C  storage and place on dry ice until ready. 

SECTION: preparation for 10x loading
50. Resuspend pellet in 100 µL of pre-chilled RSB Buffer (ensure that there is no Tween-20 in this solution!) using a P200 pipette. Mix carefully and thoroughly. Add 200 µL more of RSB using a P1000 pipette. Mix carefully and thoroughly. Total volume should be 300 µL .

SECTION: Fixation and Permeabilization
22. Add 1 mL  of FA-NT solution using a P1000 pipette, mixing the first 1 mL  carefully and thoroughly with the nuclei suspension. Add 2 mL  more of FA-NT solution. The total volume in the tube should be 4 mL. 

SECTION: Fixation and Permeabilization
23. Immediately spike 3 µL of glacial acetic acid to nuclei suspension.

SECTION: Nuclei Extraction
6. Dounce with pestle "A" until resistance subsides  (~40 strokes) on ice .

None. Pre-chill all Douncers and pestle
SECTION: Nuclei Extraction
5. Place sample into a clean, pre-chilled mortar filled with 2 mL of EZ Lysis Buffer.
SECTION: Nuclei Extraction
7. Place pestle "A" in 50mL Falcon tube to hold until ready for cleaning.
SECTION: Nuclei Extraction
8. Douce with pestle "B" until resistance subsides (~40 strokes) on ice .

SECTION: Nuclei Extraction
9. Place pestle "B" in 50mL Falcon tube to hold until ready for cleaning.
SECTION: Nuclei Extraction
10. Transfer the 2 mL homogenate to a pre-chilled 15mL Falcon Tube.

SECTION: Nuclei Extraction
11. Add 3 mL of EZ Lysis Buffer to raise the sample volume to 5 mL total -- volumes can be added to wash out the mortar before being added, to maximize nuclei transfer. 

SECTION: Nuclei Extraction
12. Incubate for 5 min   on ice .


SECTION: Nuclei Extraction
12.1. During this incubation, spray mortars and pestles with 10% bleach, let sit, and rinse with distilled water in order to clean off remaining tissue. After rinsing, soak mortars and pestles in 10% bleach and store until next use.  
SECTION: Nuclei Extraction
13. Spin down nuclei at 500 x g, 5 min, 4 °C in pre-cooled swinging bucket centrifuge.

SECTION: Nuclei Extraction
14. Carefully remove and discard supernatent. 
SECTION: Nuclei Extraction
15. Resuspend pellet in 1 mL of EZ Lysis Buffer using a P1000 pipette. Mix carefully and thoroughly. Add another 
4 mL of EZ Lysis Buffer. Mix carefully and thoroughly. The total volume should be 5 mL . 

SECTION: Nuclei Extraction
16. Incubate for 5 min   on ice .
SECTION: Nuclei Extraction
17. Spin down nuclei at 500 x g, 5 min, 4 °C in pre-cooled swinging bucket centrifuge.

SECTION: preparation for 10x loading
51. Filter through a   filter into a pre-chilled FACS tube.

SECTION: Nuclei Extraction
18. Carefully remove and discard supernatent. 
SECTION: Nuclei Extraction
19. Resuspend pellet in 1 mL of RSB Buffer.

SECTION: Nuclei Extraction
20. Filter 1 mL of nuclei suspension through a pre-chilled    filter cap FACS tubes.
SECTION: Fixation and Permeabilization
25. Immediately after, quench the fixation reaction by adding 3 µL of 1 M Glycine. Use P1000 pipette to mix sample thoroughly to ensure equal distribution of glycine throughout solution.

SECTION: Fixation and Permeabilization
26. Filter through a   filter into new, pre-chilled 15mL Falcon Tube.

SECTION: Fixation and Permeabilization
27. Spin down nuclei at 850 x g, 5 min, 4 °C .

SECTION: Fixation and Permeabilization
28. Remove and discard supernatent. 
SECTION: Primary Antibody Stain
30. Incubate for 15 min at 4 °C while rocking.

SECTION: Primary Antibody Stain
31. Spin down nuclei at 850 x g, 5 min, 4 °C .

SECTION: Primary Antibody Stain
32. Remove and discard supernatent. 
SECTION: Primary Antibody Stain
33. Resuspend pellet in 200 µL of primary antibody diluted to the appropriate concentration in Blocking Buffer.
SECTION: Primary Antibody Stain
34. Incubate for 60 min at 4 °C while rocking.

SECTION: Primary Antibody Stain
35. Spin down nuclei at 850 x g, 5 min, 4 °C .
SECTION: Primary Antibody Stain
36. Carefully remove and discard supernatent.
SECTION: Primary Antibody Stain
37. Resuspend pellet in 500 µL of PBST.

SECTION: Primary Antibody Stain
38. Incubate 5 min  on ice .

SECTION: Primary Antibody Stain
40. Spin down nuclei at 850 x g, 5 min, 4 °C .

SECTION: Primary Antibody Stain
39. Repeat wash steps 34-37 to  to ensure removal of any excess antibodies.
SECTION: [OPTIONAL] Secondary Antibody Staining
42. Resuspend pellet in 200 µL of secondary antibody diluted 1:1000 in Blocking Buffer.

SECTION: Primary Antibody Stain
41. Remove and discard supernatent. If preforming a secondary antibody stain, proceed to step 42. If loading onto 10x, proceed to step 50.
SECTION: [OPTIONAL] Secondary Antibody Staining
43. Incubate for 20 min at 4 °C while rocking.
SECTION: [OPTIONAL] Secondary Antibody Staining
44. After incubation, spike in 2 µL of 100x DAPI into each sample. Mix carefully and thoroughly using a P200 pipette.

SECTION: [OPTIONAL] Secondary Antibody Staining
45. Repeat wash steps 34-37 two times to ensure removal of any excess antibodies in solution.
SECTION: [OPTIONAL] Secondary Antibody Staining
46. Spin down nuclei at 850 x g, 5 min, 4 °C .
SECTION: [OPTIONAL] Secondary Antibody Staining
47. Resuspend pellet in 500 µL of PBST.

SECTION: [OPTIONAL] Secondary Antibody Staining
48. filter through a 20  filter into a pre-chilled FACS tube.

SECTION: [OPTIONAL] Secondary Antibody Staining
49. Keep samples in dark on ice  until processing via flow cytometry.

SECTION: preparation for 10x loading
52. Count nuclei using hemocytometer chamber. Keep nuclei on ice until ready for 10X loading. 

SECTION: Before you start the protocol
1. All steps should be performed on ice or at 4 °C . Cool a swinging bucket centrifuge to 4 °C. Prepare all reagents and chill on ice . 

SECTION: Before you start the protocol
2. Clean one set of douncers (mortar, pestle A and pestle B) for every sample as follows. Make sure to thoroughly wash inside the mortar and at the end of each pestle where the sample will come in contact:
        Rinse with distilled water. 
        Spray with 70% ethanol. Let sit for ~1 min 
        Rinse with distilled water
        Spray with RNase Zap. Let sit for 1 min 
        Rinse with distilled water
        Rinse with DNase and RNase free double distilled water
        Let air dry on kimewipe

Once dry, pre-chill on on ice . 

SECTION: Before you start the protocol
3. Pre-chill all tubes on ice . For each sample, you will need:
        3 x 15mL Falcon Tubes
        1 x 1.5mL Eppendorf tube
        2 x 35µm filter-cap FACS tubes
        2 x 20µm filters