Multi tissue processing for single cell sequencing of human immune cells

No processing, go straight to ficoll layering.

PlaceOn iceuntil other tissues have caught up.

Mash the lymphoid tissue through a 70 µm filter placed on top of a 50mL falcon, using the plunger from a 2mLsyringe as a pestle.

Occasionally wash the filter with x-vivo + 1% FBS as you mash the tissue.

Depending on the size of the tissue, top up the filtered cell suspension to 30mL to 50mLwith x-vivo + 1% FBS.

Place On iceuntil other tissues have caught up.

We receive around 5g of tissue and the protocol will need to be scaled up if more tissue is being processed.

Chop up the tissue with scissors into 0.5 cm pieces.

Do not overload the Gentlemacs C-tube, with no more than 2.5gof tissue.

Transfer to Gentlemacs tube and add 2.5mL of collagenase and 2.5mLx-vivo.

Run the following programme that takes 0h 32m 0s .

Loop .

• Loop .(1/3)
• Loop. (2/3)
• Loop. (3/3)

Spin 50rpm.

Spin 350rpm.

Ramp900rpm,0h 0m 0s 0h 0m 12s .

Spin 700rpm .

Ramp 1000rpm .

Spin 1500rpm .

Spin 1900rpm.

Spin 1500rpm .

Spin 1900rpm.

Temperature on 37°C and loop.

• loop (1/2).
• loop (2/2).

Add 20µL of 0.5millimolar (mM) EDTA ( 2millimolar (mM) final conc.) per5mLof collagenase to neutralise and shake to mix.

Pour and scrape digested tissue into a 70 µm cell strainer placed on top of a 50mL falcon.

Use the plunger of a 2mL syringe to mash tissue through the filter, like a pestle.

Occasionally wash the filter with x-vivo + 1% FBS as you mash the tissue.

Depending on the size of the tissue, top up the filtered cell suspension to 30mL to 50mLwith x-vivo + 1% FBS.

Place On ice until other tissues have caught up.

Wash jejunum with PBS + 0.04% BSA to remove any chime.

Chop up the jejunum with scissors into 0.5 cm pieces.

Transfer to a 50mL falcon tube and add 10mL of x-vivo + 2millimolar (mM) DTT +5millimolar (mM) EDTA + 1% FBS and put in the 37°Cincubator for 0h 20m 0s and shake after 0h 10m 0s.

Put jejunum chemical digest through a 70 µm filter on top of a 50mLfalcon and rinse with 10mLof x-vivo + 1% FCS.

The wash through from the filter contains the IEL cells, keeping the falcon 37On ice.

Scrape tissue from the filter back into a 50mLfalcon and repeat the digest with 10mL x-vivo + 2millimolar (mM) DTT +5millimolar (mM) EDTA + 1% FBS and place back in the 37°Cincubator for 0h 20m 0s, and shake after 0h 10m 0s.

Put jejunum digest through a 70 µm filter on top of the 50mLfalcon containing the IEL cells and rinse with 10mLof x-vivo + 1% FCS. Keep the IEL cells37On ice.

Scrape tissue from the filter into a Gentlemacs C tube and digest with 2.5mL of collagenase and 2.5mLof x-vivo and run the programme called ‘Sarah’ takes0h 32m 0s, with various mixing speeds.

Add 20µL of 0.5millimolar (mM) EDTA ( 2millimolar (mM) final conc) per 5mLof collagenase to neutralise and shake to mix.

Pour and scrape digested tissue into a 70 µm cell strainer placed on top of a 50mLfalcon.

Use the plunger of a 2mL syringe to mash tissue through the filter, like a pestle.

Occasionally wash the filter with x-vivo + 1% FBS as you mash the tissue, cells that pass through the filter are LP cells .

Depending on the size of the tissue, top up the filtered cell suspension to 30mLto 50mL with x-vivo + 1% FBS.

Place 37On ice until other tissues have caught up.

Protocol from Haniffa Lab, Newcastle University https://www.protocols.io/view/human-skin-single-cell-dissociation-ripd4dn

Chop into ~0.5 cm2 sized pieces. Remove as much dermis from each as possible using a razor blade - be careful, extremely sharp. Discard the dermis layer.

Incubate the retained skin in dispase for 2h 0m 0s to 3h 0m 0s at 37°C, to allow the epidermis to be stripped.

Separate the epidermis from the dermis using fine forceps. These can be kept separate or processed together.

Wash in PBS.

Add collagenase at 3X the volume of the tissue and incubate at 37°C 3h 0m 0s.

Add 20µL of 0.5millimolar (mM) EDTA (2millimolar (mM) final conc) per 5mL of collagenase to neutralise and shake to mix.

Scrape the digested skin and media into a70 µm filter on top of a 50mL falcon.

Use the plunger from a2mL syringe to mash the skin through the filter, like a pestle.

Occasionally wash the filter with x-vivo + 1% FBS as you mash the tissue.

Depending on the size of the tissue, top up the filtered cell suspension to 30mL to 50mL with x-vivo + 1% FBS.

Place 37On ice until other tissues have caught up, or if processing the next day alone proceed with cell count and ficoll.

Once all the tissues have reached a cell suspension, spin at 600x g.

Pour off supernatant and resuspend in x-vivo + 1% FBS, the volume to resuspend depends what you are going to layer over ficoll.

There is no exact science to the layering but as a guide:

a. Spleen - `90mL`



b. Lymph nodes - `7mL`



c. Non-lymphoid tissue - up to`60mL` 



d. Skin - `7mL`

Bone marrow 10mL + 20mL x-vivo layer on 15mL ficoll per 50mL falcon.

Blood up to 15mL +15mL x-vivo layer on 15mL ficoll per 50mL falcon.

Spleen 30mL cells suspension over 15mL ficoll per 50mL falcon x3.

Lymph nodes 7mLcells suspension over 8mL ficoll per 15mL falcon.

Non-lymphoid tissue depending on the size of the cell pellet up to30mL cell suspension over 5mLficoll x2.

Skin 7mL cells suspension over8mL ficoll per 15mL falcon.

Spin tubes at 400x g with slow deceleration. Takes around 0h 40m 0s to run.

In a 15mL falcon, add from0.5mL to 3mL of sample (up to 5 million cells).

Add 25µL of CD66b positive selection cocktail, mix and incubate for 0h 3m 0s at 37Room temperature.

Vortex RapidSpheres for 0h 0m 30s.

Add 25µLof RapidSpheres, mix and incubate for 0h 3m 0s at 37Room temperature.

Add25µL of RBC depletion reagent per 1mLof sample and mix.

Immediately place the samples on a magnet for 0h 5m 0s.

Carefully pipette off the supernatant to a fresh tube and place 37On ice.

Wash the beads with 5mL of PBS 1% FBS + 1millimolar (mM) EDTA and place back on the magnet for 0h 5m 0s.

Collect supernatant and add to the fresh tube in step 60 .

Throw away the leftover tube with beads as this contains the granulocytes and RBC.

Count cells from each tissue after ficoll (and CD66b / RBC depletion).

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.

Take at least 500k MNC per tissue (use 750k to 1 million cells if available) into a1.5mL lo-bind eppendorf.

Spin cells at 600x g, remove as much supernatant as possible and resuspend in 50µLPBS+0.04% BSA.

Record which hashtag is used for which tissue.

Add 5µL FC block and incubate at 4°C for 0h 10m 0s.

Spin each hashtag at 14000x g.

Add 0.5µLof hashtag to each tube.

Incubate at 4°C for 0h 30m 0s.

Make up lyophilised CITE-Seq antibodies (see - section CITE-Seq section )

Top up to 500µLwith PBS + 0.04% BSA, and spin at 600x g, and remove supernatant.

Wash cells with 500µLwith PBS + 0.04% BSA, and spin at 600x g, and remove supernatant.

Resuspend in 100µLof PBS + 0.04% BSA.

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.

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.

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 1.5mL lo-bind eppendorf.

Ideally you want 300k - 400k from each tissue. Record the total volume.

Flick to mix the cells really well.

Remove ⅓ of the cell volume to a new 1.5mL tube and label as Unstim and top up to 500µL with PBS + 0.04% BSA. Spin at600x g and proceed to the CITE-Seq section .

To the remaining ⅔ of pooled MNC, label the tube as Stim and top up to 1mL with x-vivo + 1% FCS and proceed to MNC stimulation .

Get the MNC stim on as it takes2h 0m 0s.

Pool culture in MNC in1mL of x-vivo + 1% FBS for 2h 0m 0s at 37°C with 2µLof cell stim cocktail (PMA+I). Flick tube to mix cells every 0h 30m 0s to 0h 40m 0s.

Culture MNC at no more than 2 million cells per ml.

Incubate for 2h 0m 0s , move to Cite-Seq of stimulated cells.

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

Spin lyophilised reagent at 10000x g.

Add 27.5µL Cell staining buffer to the lyophilised CITE-Seq reagent and briefly vortex.

Incubate at 37Room temperature for 0h 5m 0s.

Briefly vortex again, then spin at 10000x g.

Transfer entire volume to a lo-bind PROTEIN tube.

Spin at 14000x g,4°C.

Store in the fridge until ready to use.

Spin the unstim pool MNC at 600x gand remove supernatant.

Resuspend cells in 50µL of PBS + 0.04% BSA.

No need to add FC block, as already done at hashtag stage.

If not hashtagged already, then add 5µLFC block for 0h 10m 0s at 4°C.

Add10µLof CITE-Seq 130Ab and incubate at 4°C for 0h 30m 0s.

(Take 10x reagent out of the freezer to warm up to , during CITE-Seq incubation. It takes to warm up to .) 4Room temperature, during CITE-Seq incubation. It takes 0h 30m 0s to warm up to 4Room temperature.)

Top up to500µLwith PBS + 0.04% BSA, and spin at 600x g, and remove supernatant.

Wash cells with 500µLwith PBS + 0.04% BSA, and spin at 600x g, and remove supernatant.

Resuspend cells in 250µL PBS + 0.04% BSA and put through a flowmi filter. Rinse out 1.5mL tube with 250µLPBS + 0.04% BSA, and put this through the same Flowmi filter.

Spin at 600x g, and remove supernatant.

Resuspend in 100µL of PBS + 0.04% BSA.

Count the unstim pooedl MNC sample.

Make sure cells are well mixed and count with trypan blue.

2µL of cells to 8µL 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.

Load cells at 1,000 cells per 1µL (Max 2,000 cells / µl).

Dilute the sample (if needed).

Load 15,000 cells per tissue, 30,000 cells per 10x GEM reaction.

After the 2h 0m 0s stimulation, spin the stim pool MNC at 600x g and remove supernatant.

Resuspend cells in 15µL of PBS + 0.04% BSA.

Add 12.5µLof CITE-Seq 130Ab and incubate at 4°C for 0h 30m 0s.

( Take 10x reagent out of the freezer to warm up to , during CITE-Seq incubation. It takes to warm up to 4Room temperature, during CITE-Seq incubation. It takes 0h 30m 0s to warm up to4Room temperature.)

Top up to 500µL with PBS + 0.04% BSA, and spin at 600x g, and remove supernatant.

Wash cells with 500µL with PBS + 0.04% BSA, and spin at 600rpm, and remove supernatant.

Resuspend cells in 250µL PBS + 0.04% BSA and put through a flowmi filter. Rinse out 1.5mL tube with 250µLPBS + 0.04% BSA, and put this through the same Flowmi filter.

Spin at 600x g, and remove supernatant.

Resuspend in 100µL of PBS + 0.04% BSA.

Count the Stim pooled MNC sample.

Make sure cells are well mixed and count with trypan blue.

2µLof cells to 8µLof Trypan blue. If count all 25 squares of the haemocytometer, then:

Cell count x 5 x 0.1 x 10,000 = Total cell count.

Load cells at 1,000 cells per 1µL (Max 2,000 cells / µl).

Dilute the sample (if needed) in PBS + 0.04% BSA.

Load 15,000 cells per tissue, 30,000 cells per 10x GEM reaction.

When all the 10x GEMs have been processed and they look good, pellet any leftover pooled unstim or stim MNC at 600rpm and take off supernatant.

Flick to resuspend dry pellet and resuspend in 350µL of Qiagen RLT buffer.

Quickly vortex and freeze at -80°C until ready to extract the RNA.

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.

Fix cells and store at 4°C until they can be analysed.

Spin cells at 600x g, and remove as much supernatant as possible.

Flick to resuspend cell pellet.

Add cell freezing media dropwise, until ~ 10 million cells per ml.

Flick to mix, and transfer to labelled NUNC tubes.

Put NUNC tubes in a Mr Frosty and store at-80°C 0h 30m 0s.

Next day, transfer to LN2 storage.