TITLE: CGAP Human Spleen Dissociation, Tissue Stability Study
AUTHORS: Anna Wilbrey-Clark, Adam Hunter

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Mechanical dissociation of human spleen tissue to single cells for use in 10X chromium scRNA-Seq preparation. </div></div>

[STEPS]
?. Receive spleen punch in hypothermasol FRS (Sigma H4416) solution.
?. Place spleen punch onto 100mm Glass Petri Dish.
?. Add 10ml Cold PBS.
?. Slice spleen punch into pieces (less than 10x10x10mm).
?. Transfer spleen pieces and PBS into 50ml Falcon Tube.
?. Add 100µl DNAse I dropwise to spleen punch in PBS to a final concentration of 100µg/ml (DNase treatment is not essential for spleen dissociation).
?. Incubate at room temperature for 5 min.
?. Mash spleen through 100µM cell strainer above a 50ml Falcon Tube using a 2.0ml syringe plunger, washing through with 10ml Cold PBS.
?. Transfer to 15ml Falcon Tube.
?. Centrifuge at 500g for 5 min at 4°C.
?. Add 1ml Red Cell Lysis buffer to the pellet and resuspend by racking/tapping.
?. Place on ice 5 min with periodic agitation.
?. Add 9ml Cold PBS.
?. Centrifuge at 500g for 5 min at 4°C.
?. Resuspend pellet in 5ml Cold PBS with 0.04% BSA.
?. Filter through a 100µM cell strainer above a 50ml Falcon Tube.
?. Wash cell strainer through with 5 ml Cold PBS with 0.04% BSA.
?. Count cells using trypan blue and a C-Chip (manually).
?. Remove dead cells by following “MACS Live Dead Separation”.
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?. Dilute cells to 1x106 cell per ml in 0.04% BSA and proceed to 10X preparation for scRNA sequencing.
?. Ensure all unused tissue, equipment and tubes that have been in contact with primary tissue are placed into Virkon in sweetie jar for a minimum of 1 hour. After this time aspirate and disposing in relevant sharps or waste routes.
?. Perform Freezing Human Tissue in Isopentane before beginning Dissociation Protocol, if frozen samples are also to be collected e.g. for bulk RNA / DNA extraction etc.
?. ABC1Material
Quantity
Supplier Info
2100mm Petri Dish
1
Corning (430591)
3Cold PBS
30ml
GIBCO (14190-144)
4Forceps
2
ThermoFisher UK Ltd (15232290)
5Scalpel
1
Swann-Morton Ltd (0507)
6DNAse I (Stock solution 10mg/ml)
100ul
Sigma (11284932001)
7100uM Cell Strainers
2
ThermoFisher UK Ltd (15380801)
850ml Falcon Tubes
2
Falcon (352098)
92.0ml Syringe
2
ThermoFisher UK Ltd. (10673555)
1015ml Falcon Tubes
2
Falcon (352097)
11Red Cell Lysis Buffer
5ml
Life Technologies Ltd. (00-4333-57)
12BSA
400ul
Sigma Aldrich (A7906-10G)
130.5ml Eppendorf
1
Eppendorf (0030 108.035)
14Trypan Blue
20ul
Fisher Scientific (11414815)
15C-Chips
1
Cambridge Bioscience (DHC-N01-50)
ABC1Material
Quantity
Supplier Info
2100mm Petri Dish
1
Corning (430591)
3Cold PBS
30ml
GIBCO (14190-144)
4Forceps
2
ThermoFisher UK Ltd (15232290)
5Scalpel
1
Swann-Morton Ltd (0507)
6DNAse I (Stock solution 10mg/ml)
100ul
Sigma (11284932001)
7100uM Cell Strainers
2
ThermoFisher UK Ltd (15380801)
850ml Falcon Tubes
2
Falcon (352098)
92.0ml Syringe
2
ThermoFisher UK Ltd. (10673555)
1015ml Falcon Tubes
2
Falcon (352097)
11Red Cell Lysis Buffer
5ml
Life Technologies Ltd. (00-4333-57)
12BSA
400ul
Sigma Aldrich (A7906-10G)
130.5ml Eppendorf
1
Eppendorf (0030 108.035)
14Trypan Blue
20ul
Fisher Scientific (11414815)
15C-Chips
1
Cambridge Bioscience (DHC-N01-50)