TITLE: Multi tissue processing for single cell sequencing of human immune cells
AUTHORS: Daniel  Rainbow, Sarah Howlett, Lorna Jarvis, Joanne Jones

[DESCRIPTION]
This protocol has been developed for the simultaneous processing of multiple human tissues to extract immune cells for single cell RNA sequencing using the 10X platform, and ideal for atlasing projects. Included in this protocol are the steps needed to go from tissue to loading the 10X Chromium for single cell RNA sequencing and includes the hashtag and CiteSeq labelling of cells as well as the details needed to stimulate cells with PMA+I.

[GUIDELINES]
This protocol has been optimised for extracting immune cells from small pieces of tissue (around 10g or less) and has divided tissues into five categories depending on how much mechanical or chemical digestion is needed to enable extraction of immune cells, as shown in the workflow diagram. Blood and bone marrow need no processing. Lymphoid tissues like spleen and lymph nodes require a gentle mashing to make a cell suspension. Non-lymphoid tissues like lung, liver and kidney require both a mechanical and collagenase digestion. The gut and skin require more specialised protocols and use published protocols from the James and Hanifa laboratories. 
We include an activation step using PMA+I for 2 hours, however this stimulation condition will need optimising depending on the hypothesis being tested.

  


[STEPS]
SECTION: Tissue to cell suspension - Bone Marrow and Blood
1. No processing, go straight to ficoll layering.
SECTION: Tissue to cell suspension - Bone Marrow and Blood
2. Placeon iceuntil other tissues have caught up.

SECTION: Tissue to cell suspension - Lymphoid Tissues (Spleen, Lymph node)
3. Mash the lymphoid tissue through a   filter placed on top of a 50 mL falcon, using the plunger from a 2 mLsyringe as a pestle.

SECTION: Tissue to cell suspension - Lymphoid Tissues (Spleen, Lymph node)
4. Occasionally wash the filter with x-vivo + 1% FBS as you mash the tissue.
SECTION: Tissue to cell suspension - Lymphoid Tissues (Spleen, Lymph node)
5. Depending on the size of the tissue, top up the filtered cell suspension to 30 mL to 50 mLwith x-vivo + 1% FBS.

SECTION: Tissue to cell suspension - Lymphoid Tissues (Spleen, Lymph node)
6. Place on iceuntil other tissues have caught up.

SECTION: Tissue to cell suspension - Non-lymphoid tissue (Lung, Liver, Kidney)
7. We receive around 5 g of tissue and the protocol will need to be scaled up if more tissue is being processed.
 

SECTION: Tissue to cell suspension - Non-lymphoid tissue (Lung, Liver, Kidney)
8. Chop up the tissue with scissors into   pieces.

SECTION: Tissue to cell suspension - Non-lymphoid tissue (Lung, Liver, Kidney)
9. Do not overload the Gentlemacs C-tube, with no more than 2.5 gof tissue.

SECTION: Tissue to cell suspension - Non-lymphoid tissue (Lung, Liver, Kidney)
10. Transfer to Gentlemacs tube and add 2.5 mL of collagenase and 2.5 mLx-vivo.

SECTION: Tissue to cell suspension - Non-lymphoid tissue (Lung, Liver, Kidney)
11. Run the following programme that takes 32 min . 
SECTION: Tissue to cell suspension - Non-lymphoid tissue (Lung, Liver, Kidney)
12. Add 20 µL of 0.5 millimolar (mM) EDTA ( 2 millimolar (mM) final conc.) per5 mLof collagenase to neutralise and shake to mix.

SECTION: Tissue to cell suspension - Non-lymphoid tissue (Lung, Liver, Kidney)
13. Pour and scrape digested tissue into a   cell strainer placed on top of a 50 mL falcon. 

SECTION: Tissue to cell suspension - Non-lymphoid tissue (Lung, Liver, Kidney)
14. Use the plunger of a 2 mL syringe to mash tissue through the filter, like a pestle.

SECTION: Tissue to cell suspension - Non-lymphoid tissue (Lung, Liver, Kidney)
15. Occasionally wash the filter with x-vivo + 1% FBS as you mash the tissue.
SECTION: Tissue to cell suspension - Non-lymphoid tissue (Lung, Liver, Kidney)
16. Depending on the size of the tissue, top up the filtered cell suspension to 30 mL to 50 mLwith x-vivo + 1% FBS.

SECTION: Tissue to cell suspension - Non-lymphoid tissue (Lung, Liver, Kidney)
17. Place on ice until other tissues have caught up.

SECTION: Tissue to cell suspension - Non-lymphoid tissue (Lung, Liver, Kidney)
11.1. Loop .
Loop .(1/3)
Loop. (2/3)
Loop. (3/3)
SECTION: Tissue to cell suspension - Non-lymphoid tissue (Lung, Liver, Kidney)
11.2. Ramp900 rpm 12 s .

SECTION: Tissue to cell suspension - Non-lymphoid tissue (Lung, Liver, Kidney)
11.3. Spin 700 rpm, 1 s .

SECTION: Tissue to cell suspension - Non-lymphoid tissue (Lung, Liver, Kidney)
11.4. Ramp 1000 rpm, 8 s .

SECTION: Tissue to cell suspension - Non-lymphoid tissue (Lung, Liver, Kidney)
11.5. Spin 1500 rpm, 1 s .

SECTION: Tissue to cell suspension - Non-lymphoid tissue (Lung, Liver, Kidney)
11.6. Spin 1900 rpm, 4 s.

SECTION: Tissue to cell suspension - Non-lymphoid tissue (Lung, Liver, Kidney)
11.7.  Spin 1500 rpm, 1 s .

SECTION: Tissue to cell suspension - Non-lymphoid tissue (Lung, Liver, Kidney)
11.8. Spin 1900 rpm, 3 s.

SECTION: Tissue to cell suspension - Non-lymphoid tissue (Lung, Liver, Kidney)
11.9. Temperature on 37 °C and loop.

loop (1/2).
loop (2/2).
SECTION: Tissue to cell suspension - Non-lymphoid tissue (Lung, Liver, Kidney)
11.10. Spin 50 rpm, 15 min.

SECTION: Tissue to cell suspension - Non-lymphoid tissue (Lung, Liver, Kidney)
11.11. Spin 350 rpm, 20 s.

SECTION: Tissue to cell suspension - Jejunum
18. 
 
Wash jejunum with PBS + 0.04% BSA to remove any chime.
SECTION: Tissue to cell suspension - Jejunum
19. Chop up the jejunum with scissors into  pieces.

SECTION: Tissue to cell suspension - Jejunum
20. Transfer to a 50 mL falcon tube and add 10 mL of x-vivo + 2 millimolar (mM) DTT +5 millimolar (mM) EDTA + 1% FBS and put in the 37 °Cincubator for 20 min and shake after 10 min.

SECTION: Tissue to cell suspension - Jejunum
21. Put jejunum chemical digest through a   filter on top of a 50 mLfalcon and rinse with 10 mLof x-vivo + 1% FCS.

SECTION: Tissue to cell suspension - Jejunum
22. The wash through from the filter contains the IEL cells, keeping the falcon on ice.

SECTION: Tissue to cell suspension - Jejunum
23. Scrape tissue from the filter back into a 50 mLfalcon and repeat the digest with 10 mL x-vivo + 2 millimolar (mM) DTT +5 millimolar (mM) EDTA + 1% FBS and place back in the 37 °Cincubator for 20 min, and shake after 10 min.

SECTION: Tissue to cell suspension - Jejunum
24. Put jejunum digest through a   filter on top of the 50 mLfalcon containing the IEL cells and rinse with 10 mLof x-vivo + 1% FCS. Keep the IEL cellson ice.

SECTION: Tissue to cell suspension - Jejunum
25. Scrape tissue from the filter into a Gentlemacs C tube and digest with 2.5 mL of collagenase and 2.5 mLof x-vivo and run the programme called ‘Sarah’ takes32 min, with various mixing speeds.

SECTION: Tissue to cell suspension - Jejunum
26. Add 20 µL of 0.5 millimolar (mM) EDTA ( 2 millimolar (mM) final conc) per 5 mLof collagenase to neutralise and shake to mix.

SECTION: Tissue to cell suspension - Jejunum
27. Pour and scrape digested tissue into a   cell strainer placed on top of a 50 mLfalcon.

SECTION: Tissue to cell suspension - Jejunum
28. Use the plunger of a 2 mL syringe to mash tissue through the filter, like a pestle.

SECTION: Tissue to cell suspension - Jejunum
29. Occasionally wash the filter with x-vivo + 1% FBS as you mash the tissue, cells that pass through the filter are LP cells.
SECTION: Tissue to cell suspension - Jejunum
30. Depending on the size of the tissue, top up the filtered cell suspension to 30 mLto 50 mL with x-vivo + 1% FBS.

SECTION: Tissue to cell suspension - Jejunum
31. Place on ice until other tissues have caught up.

SECTION: Tissue to cell suspension - Skin
32. Protocol from Haniffa Lab, Newcastle University https://www.protocols.io/view/human-skin-single-cell-dissociation-ripd4dn
 

Chop into ~  sized pieces. Remove as much dermis from each as possible using a razor blade - be careful, extremely sharp. Discard the dermis layer.

SECTION: Tissue to cell suspension - Skin
33. Incubate the retained skin in dispase for 120 min to 180 min at 37 °C, to allow the epidermis to be stripped.

SECTION: Tissue to cell suspension - Skin
34. Separate the epidermis from the dermis using fine forceps. These can be kept separate or processed together.
SECTION: Tissue to cell suspension - Skin
35. Wash in PBS.
SECTION: Tissue to cell suspension - Skin
36. Add collagenase at 3X the volume of the tissue and incubate at 37 °C 180 min.

SECTION: Tissue to cell suspension - Skin
37. Add 20 µL of 0.5 millimolar (mM) EDTA (2 millimolar (mM) final conc) per 5 mL of collagenase to neutralise and shake to mix.

SECTION: Tissue to cell suspension - Skin
38. Scrape the digested skin and media into a  filter on top of a 50 mL falcon.

SECTION: Tissue to cell suspension - Skin
39. Use the plunger from a2 mL syringe to mash the skin through the filter, like a pestle.

SECTION: Tissue to cell suspension - Skin
40. Occasionally wash the filter with x-vivo + 1% FBS as you mash the tissue.
SECTION: Tissue to cell suspension - Skin
41. Depending on the size of the tissue, top up the filtered cell suspension to 30 mL to 50 mL with x-vivo + 1% FBS.

SECTION: Tissue to cell suspension - Skin
42. Place on ice until other tissues have caught up, or if processing the next day alone proceed with cell count and ficoll.

SECTION: Cell suspension to MNC  - Wash cell suspension
43. Once all the tissues have reached a cell suspension, spin at 600 x g, 10 min.

SECTION: Cell suspension to MNC  - Wash cell suspension
44. Pour off supernatant and resuspend in x-vivo + 1% FBS, the volume to resuspend depends what you are going to layer over ficoll.
SECTION: Cell suspension to MNC  - Wash cell suspension
45. There is no exact science to the layering but as a guide:
    a. Spleen - 90 mL
    b. Lymph nodes - 7 mL
    c. Non-lymphoid tissue - up to60 mL 
    d. Skin - 7 mL

SECTION: Cell suspension to MNC - Ficoll
46. 
 
Bone marrow 10 mL + 20 mL x-vivo layer on 15 mL ficoll per 50 mL falcon. 

SECTION: Cell suspension to MNC - Ficoll
47.  Blood up to 15 mL +15 mL x-vivo layer on 15 mL ficoll per 50 mL falcon. 

SECTION: Cell suspension to MNC - Ficoll
48.  Spleen 30 mL cells suspension over 15 mL ficoll per 50 mL falcon x3.

SECTION: Cell suspension to MNC - Ficoll
49.  Lymph nodes 7 mLcells suspension over 8 mL ficoll per 15 mL falcon.

SECTION: Cell suspension to MNC - Ficoll
50. Non-lymphoid tissue depending on the size of the cell pellet up to30 mL cell suspension over 5 mLficoll x2.

SECTION: Cell suspension to MNC - Ficoll
51.  Skin 7 mL cells suspension over8 mL ficoll per 15 mL falcon. 

SECTION: Cell suspension to MNC - Ficoll
52. Spin tubes at 400 x g, 25 min with slow deceleration. Takes around 40 min to run.

SECTION: Cell suspension to MNC - CD66b and RBC depletion
53. 
 
In a 15 mL falcon, add from0.5 mL to 3 mL of sample (up to 5 million cells).

SECTION: Cell suspension to MNC - CD66b and RBC depletion
54.  Add 25 µL of CD66b positive selection cocktail, mix and incubate for 3 min at 37 Room temperature.

SECTION: Cell suspension to MNC - CD66b and RBC depletion
55.  Vortex RapidSpheres for 30 s. 

SECTION: Cell suspension to MNC - CD66b and RBC depletion
56. Add 25 µLof RapidSpheres, mix and incubate for 3 min at 37 Room temperature. 

SECTION: Cell suspension to MNC - CD66b and RBC depletion
57. Add25 µL of RBC depletion reagent per 1 mLof sample and mix. 

SECTION: Cell suspension to MNC - CD66b and RBC depletion
58. Immediately place the samples on a magnet for 5 min. 

SECTION: Cell suspension to MNC - CD66b and RBC depletion
59. Carefully pipette off the supernatant to a fresh tube and place on ice. 
 


SECTION: Cell suspension to MNC - CD66b and RBC depletion
60.  Wash the beads with 5 mL of PBS 1% FBS + 1 millimolar (mM) EDTA and place back on the magnet for 5 min.

SECTION: Cell suspension to MNC - CD66b and RBC depletion
61. Collect supernatant and add to the fresh tube in step 60.
SECTION: Cell suspension to MNC - CD66b and RBC depletion
62.  Throw away the leftover tube with beads as this contains the granulocytes and RBC.


SECTION: Hashtag, CITE-Seq and stimulation - Hashtag
65. Take at least 500k MNC per tissue (use 750k to 1 million cells if available) into a1.5 mL lo-bind eppendorf.

SECTION: Hashtag, CITE-Seq and stimulation - Hashtag
66. Spin cells at 600 x g, 5 min, remove as much supernatant as possible and resuspend in 50 µLPBS+0.04% BSA.
SECTION: Hashtag, CITE-Seq and stimulation - Hashtag
67. Record which hashtag is used for which tissue.
SECTION: Hashtag, CITE-Seq and stimulation - Hashtag
68. Add 5 µL FC block and incubate at 4 °C for 10 min.

SECTION: Hashtag, CITE-Seq and stimulation - Hashtag
69. Spin each hashtag at 14000 x g, 10 min.

SECTION: Hashtag, CITE-Seq and stimulation - Hashtag
70. Add 0.5 µLof hashtag to each tube.

SECTION: Hashtag, CITE-Seq and stimulation - Hashtag
71. Incubate at 4 °C for 30 min.

SECTION: Hashtag, CITE-Seq and stimulation - Hashtag
72. Make up lyophilised CITE-Seq antibodies (see  - section CITE-Seq section)
SECTION: Hashtag, CITE-Seq and stimulation - Hashtag
73. Top up to 500 µLwith PBS + 0.04% BSA, and spin at 600 x g, 5 min, and remove supernatant.

SECTION: Hashtag, CITE-Seq and stimulation - Hashtag
74. Wash cells with 500 µLwith PBS + 0.04% BSA, and spin at 600 x g, 5 min, and remove supernatant.

SECTION: Hashtag, CITE-Seq and stimulation - Hashtag
75. Resuspend in 100 µLof PBS + 0.04% BSA.



SECTION: Hashtag, CITE-Seq and stimulation - Pool MNC from all tissues and split for Unstim and Stim (if required)
78. Use the post hashtag cell counts to pool MNC from each tissue at equal cell number, based on what the lowest count is, into a 1.5 mL lo-bind eppendorf.

SECTION: Hashtag, CITE-Seq and stimulation - Pool MNC from all tissues and split for Unstim and Stim (if required)
79. Ideally you want 300k - 400k from each tissue. Record the total volume.
SECTION: Hashtag, CITE-Seq and stimulation - Pool MNC from all tissues and split for Unstim and Stim (if required)
80. Flick to mix the cells really well.
SECTION: Hashtag, CITE-Seq and stimulation - Pool MNC from all tissues and split for Unstim and Stim (if required)
81. Remove ⅓ of the cell volume to a new 1.5 mL tube and label as Unstim and top up to 500 µL with PBS + 0.04% BSA. Spin at600 x g, 5 min and proceed to the CITE-Seq section.

SECTION: Hashtag, CITE-Seq and stimulation - Pool MNC from all tissues and split for Unstim and Stim (if required)
82. To the remaining ⅔ of pooled MNC, label the tube as Stim and top up to 1 mL with x-vivo + 1% FCS and proceed to MNC stimulation.

SECTION: Hashtag, CITE-Seq and stimulation - MNC Stimulation with PMA+I
83. 
 

Get the MNC stim on as it takes120 min.

SECTION: Hashtag, CITE-Seq and stimulation - MNC Stimulation with PMA+I
85. Culture MNC at no more than 2 million cells per ml. 
 

SECTION: Hashtag, CITE-Seq and stimulation - MNC Stimulation with PMA+I
86. Incubate for 120 min , move to Cite-Seq of stimulated cells.

SECTION: Hashtag, CITE-Seq and stimulation - CITE-Seq
87. Make up lyophilized CITE-Seq antibodies - each vial is enough for 500k cells, but will use 1 vial for up to 2 million cells.


SECTION: Hashtag, CITE-Seq and stimulation - CITE-Seq
88. Spin the unstim pool MNC at 600 x g, 5 minand remove supernatant.

SECTION: Hashtag, CITE-Seq and stimulation - CITE-Seq
89. Resuspend cells in 50 µL of PBS + 0.04% BSA.

SECTION: Hashtag, CITE-Seq and stimulation - CITE-Seq
90. 
No need to add FC block, as already done at hashtag stage.

SECTION: Hashtag, CITE-Seq and stimulation - CITE-Seq
91. Add10 µLof CITE-Seq 130Ab and incubate at 4 °C for 30 min. 
 

SECTION: Hashtag, CITE-Seq and stimulation - CITE-Seq
92. (Take 10x reagent out of the freezer to warm up to 4 Room temperature, during CITE-Seq incubation. It takes 30 min to warm up to 4 Room temperature.)

SECTION: Hashtag, CITE-Seq and stimulation - CITE-Seq
93. Top up to500 µLwith PBS + 0.04% BSA, and spin at 600 x g, 5 min, and remove supernatant.

SECTION: Hashtag, CITE-Seq and stimulation - CITE-Seq
94. Wash cells with 500 µLwith PBS + 0.04% BSA, and spin at 600 x g, 5 min, and remove supernatant.

SECTION: Hashtag, CITE-Seq and stimulation - CITE-Seq
95. Resuspend cells in 250 µL PBS + 0.04% BSA and put through a flowmi filter. Rinse out 1.5 mL tube with 250 µLPBS + 0.04% BSA, and put this through the same Flowmi filter. 
 

SECTION: Hashtag, CITE-Seq and stimulation - CITE-Seq
96. Spin at 600 x g, 5 min, and remove supernatant.

SECTION: Hashtag, CITE-Seq and stimulation - CITE-Seq
97. Resuspend in 100 µL of PBS + 0.04% BSA.

SECTION: Cell suspension to MNC - Count Cells
63. Count cells from each tissue after ficoll (and CD66b / RBC depletion).

SECTION: Cell suspension to MNC - Count Cells
64. Make sure cells are well mixed and count with trypan blue. If count all 25 squares of the haemocytometer, then:
Cell count x dilution factor x volume x 10,000 = Total cell count.

SECTION: Hashtag, CITE-Seq and stimulation - Count cells
76. Count cells from each tissue after the Hashtag washes as there will be cell loss, and if a particular tissue has fewer cells than needed, then repeat the hashtag process with more cells. 

SECTION: Hashtag, CITE-Seq and stimulation - Count cells
77. Make sure cells are well mixed and count with trypan blue. If count all 25 squares of the haemocytometer, then:
Cell count x dilution factor x volume x 10,000 = Total cell count.

SECTION: Hashtag, CITE-Seq and stimulation - CITE-Seq
90.1. If not hashtagged already, then add 5 µLFC block for 10 min at 4 °C.

SECTION: Hashtag, CITE-Seq and stimulation - Count cells
98. Count the unstim pooedl MNC sample.
SECTION: Hashtag, CITE-Seq and stimulation - Count cells
99. Make sure cells are well mixed and count with trypan blue.
SECTION: Hashtag, CITE-Seq and stimulation - Count cells
100. 2 µL of cells to 8 µL of Trypan blue. If count all 25 squares of the haemocytometer, then:
Cell count x 5 x 0.1 x 10,000 = Total cell count.

SECTION: Hashtag, CITE-Seq and stimulation - Load unstim for 10x
101. Load cells at 1,000 cells per 1 µL (Max 2,000 cells / µl).

SECTION: Hashtag, CITE-Seq and stimulation - Load unstim for 10x
102. Dilute the sample (if needed).
SECTION: Hashtag, CITE-Seq and stimulation - Load unstim for 10x
103. Load 15,000 cells per tissue, 30,000 cells per 10x GEM reaction.
 

SECTION: Hashtag, CITE-Seq and stimulation - Cite-Seq of stimulated cells
104. After the 120 min stimulation, spin the stim pool MNC at 600 x g, 5 min and remove supernatant.

SECTION: Hashtag, CITE-Seq and stimulation - Cite-Seq of stimulated cells
105. Resuspend cells in 15 µL of PBS + 0.04% BSA.
 


SECTION: Hashtag, CITE-Seq and stimulation - Cite-Seq of stimulated cells
106. Add 12.5 µLof CITE-Seq 130Ab and incubate at 4 °C for 30 min.

SECTION: Hashtag, CITE-Seq and stimulation - Cite-Seq of stimulated cells
107. (Take 10x reagent out of the freezer to warm up to4 Room temperature, during CITE-Seq incubation. It takes 30 min to warm up to4 Room temperature.)

SECTION: Hashtag, CITE-Seq and stimulation - Cite-Seq of stimulated cells
108. Top up to 500 µL with PBS + 0.04% BSA, and spin at 600 x g, 5 min, and remove supernatant.

SECTION: Hashtag, CITE-Seq and stimulation - Cite-Seq of stimulated cells
109. Wash cells with 500 µL with PBS + 0.04% BSA, and spin at 600 rpm, 5 min, and remove supernatant.

SECTION: Hashtag, CITE-Seq and stimulation - Cite-Seq of stimulated cells
110. Resuspend cells in 250 µL PBS + 0.04% BSA and put through a flowmi filter. Rinse out 1.5 mL tube with 250 µLPBS + 0.04% BSA, and put this through the same Flowmi filter. 
 

SECTION: Hashtag, CITE-Seq and stimulation - Cite-Seq of stimulated cells
111. Spin at 600 x g, 5 min, and remove supernatant.

SECTION: Hashtag, CITE-Seq and stimulation - Cite-Seq of stimulated cells
112. Resuspend in 100 µL of PBS + 0.04% BSA.

SECTION: Hashtag, CITE-Seq and stimulation - Count cells
113. Count the Stim pooled MNC sample.
SECTION: Hashtag, CITE-Seq and stimulation - Count cells
114. Make sure cells are well mixed and count with trypan blue.
SECTION: Hashtag, CITE-Seq and stimulation - Count cells
115. 2 µLof cells to 8 µLof Trypan blue. If count all 25 squares of the haemocytometer, then:
Cell count x 5 x 0.1 x 10,000 = Total cell count.

SECTION: Hashtag, CITE-Seq and stimulation - Load stim for 10x
116. Load cells at 1,000 cells per 1 µL (Max 2,000 cells / µl).

SECTION: Hashtag, CITE-Seq and stimulation - Load stim for 10x
117. Dilute the sample (if needed) in PBS + 0.04% BSA.
SECTION: Hashtag, CITE-Seq and stimulation - Load stim for 10x
118. Load 15,000 cells per tissue, 30,000 cells per 10x GEM reaction.
 

SECTION: Flow, Freezing and RNA from remaining cells - Remaining cells put in RLT
119. When all the 10x GEMs have been processed and they look good, pellet any leftover pooled unstim or stim MNC at 600 rpm, 5 min and take off supernatant.

SECTION: Flow, Freezing and RNA from remaining cells - Remaining cells put in RLT
120. Flick to resuspend dry pellet and resuspend in 350 µL of Qiagen RLT buffer.

SECTION: Flow, Freezing and RNA from remaining cells - Remaining cells put in RLT
121. Quickly vortex and freeze at -80 °C until ready to extract the RNA.

SECTION: Flow, Freezing and RNA from remaining cells - Flow cytometry
122. Run a flow panel to QC the sample and get proportions of the major cell types. Stain ~500k per tissue with the desired panel of antibodies. 
 

SECTION: Flow, Freezing and RNA from remaining cells - Flow cytometry
123. Fix cells and store at 4 °C until they can be analysed.

SECTION: Flow, Freezing and RNA from remaining cells - Freeze down excess cells
124. 
 
Spin cells at 600 x g, 5 min, and remove as much supernatant as possible.

SECTION: Flow, Freezing and RNA from remaining cells - Freeze down excess cells
125. Flick to resuspend cell pellet.
SECTION: Flow, Freezing and RNA from remaining cells - Freeze down excess cells
126. Add cell freezing media dropwise, until ~ 10 million cells per ml.
SECTION: Flow, Freezing and RNA from remaining cells - Freeze down excess cells
127. Flick to mix, and transfer to labelled NUNC tubes.
SECTION: Flow, Freezing and RNA from remaining cells - Freeze down excess cells
128. Put NUNC tubes in a Mr Frosty and store at-80 °C 30 min.

SECTION: Flow, Freezing and RNA from remaining cells - Freeze down excess cells
129. Next day, transfer to LN2 storage.
SECTION: Hashtag, CITE-Seq and stimulation - MNC Stimulation with PMA+I
84. Pool culture in MNC in1 mL of x-vivo + 1% FBS for 120 min at 37 °C with 2 µLof cell stim cocktail (PMA+I). Flick tube to mix cells every 30 min to 40 min.
SECTION: Hashtag, CITE-Seq and stimulation - CITE-Seq
87.1. Spin lyophilised reagent at 10000 x g, 30 s. 

SECTION: Hashtag, CITE-Seq and stimulation - CITE-Seq
87.2. Add 27.5 µL Cell staining buffer to the lyophilised CITE-Seq reagent and briefly vortex.
SECTION: Hashtag, CITE-Seq and stimulation - CITE-Seq
87.3.  Incubate at 37 Room temperature for 5 min.
SECTION: Hashtag, CITE-Seq and stimulation - CITE-Seq
87.4. Briefly vortex again, then spin at 10000 x g, 30 s. 
SECTION: Hashtag, CITE-Seq and stimulation - CITE-Seq
87.5. Transfer entire volume to a lo-bind PROTEIN tube. 
SECTION: Hashtag, CITE-Seq and stimulation - CITE-Seq
87.6. Spin at 14000 x g, 10 min, 4 °C.
SECTION: Hashtag, CITE-Seq and stimulation - CITE-Seq
87.7. Store in the fridge until ready to use.