TITLE:     Human Kidney Tumor Dissociation for single-cell genomics
AUTHORS: Evrard Bertrand, Saout Judikael, Lardenois Aurelie, Kammerer-Jacquet Solène-Florence, Rioux-Leclercq Nathalie, Chalmel Frederic

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol was developed to dissociate (clear cell) renal cell carcinoma tumors in order to study single cell genomics using the Chromium Single Cell Gene Expression technology (10X Genomics). Starting from fresh biological material, the protocol was optimized to ensure fast and reproducible processing, both of which are very important criteria in single-cell are analyzes. This protocol allowed us to obtain biologically relevant high-quality data. It can easily be adapted according to the following optional steps: i) multiple sampling in several macroscopic areas of the tumor; ii) depletion of CD45+ cell populations; iii) and, progressive freezing of the dissociated tumor cells for biobanking.</div></div>

[STEPS]
?. [Tumor Dissociation]
Material  AB1MaterialSupplier Info2Tumor Dissociation Kit, HumanMiltenyi  Biotec (130-095-929)3Gentle MACS Octo DissociatorMiltenyi  Biotec (130-096-427)4GentleMACS C tubesMiltenyi  Biotec (130-096-334)5MACS SmartStrainers 70 µmMiltenyi  Biotec (130-098-462)6RPMI 1640Thermofisher (11835030)7Surgical BladeDutscher (132522)8ForcepsDutscher (711200)950ml Falcon Tubes / 15ml Falcon TubesDutsher (352098 / 352097)101.5 ml Eppendorf tubeEppendorf (0030 108.035) 11Trypan Blue SIGMA  Aldrich (T8154)12Malassez ChamberDutscher (140501)13Petri Dish, 100 mmCorning (430591)
This core step consists of a combined mechanical and enzymatic disaggregation of the tumor tissue. It requires GentleMACS Dissociator devices.
AB1MaterialSupplier Info2Tumor Dissociation Kit, HumanMiltenyi  Biotec (130-095-929)3Gentle MACS Octo DissociatorMiltenyi  Biotec (130-096-427)4GentleMACS C tubesMiltenyi  Biotec (130-096-334)5MACS SmartStrainers 70 µmMiltenyi  Biotec (130-098-462)6RPMI 1640Thermofisher (11835030)7Surgical BladeDutscher (132522)8ForcepsDutscher (711200)950ml Falcon Tubes / 15ml Falcon TubesDutsher (352098 / 352097)101.5 ml Eppendorf tubeEppendorf (0030 108.035) 11Trypan Blue SIGMA  Aldrich (T8154)12Malassez ChamberDutscher (140501)13Petri Dish, 100 mmCorning (430591)
?. [Tumor Preparation]
Transport the fresh tumor resection on ice and collect evenly distributed punch biopsies in multiple macroscopic areas.
- Avoid all fibrous and necrotic areas. If these areas are of interest, please consider using Fluorescence Activated Cell Sorting (FACS) after dissociation to sort viable cells (not tested).
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							- Up to 9 biopsies per tumor were routinely collected (in the case of radical nephrectomies).
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?. [Tumor Dissociation]
In the petri dish, pool the punch biopsies dedicated to further dissociation.
Do not exceed 1g of total tumor tissue.
?. [Tumor Dissociation]
In the petri dish, pour 500ml Digest Mix onto the punch biopsies.
?. [Tumor Dissociation]
Run the gentle MACS Octo Dissociator Program “37_h_TDK_1”RUN
In case you own the gentleMACSTMDissociator, please successively run programs h_tumor_01, h_tumuor_02 and h_tumor_03 with 2x 30 minutes of incubation at 37°C under continuous rotation between programs 01-02 and programs 02-03.
?. [Tumor Dissociation]
Humidify the MACS SmartStrainer 70 µm with 5ml RPMI 1640 above a 50ml Falcon Tube.
?. [Tumor Dissociation]
Apply cell suspension through the strainer.
?. [Tumor Dissociation]
Centrifuge cell suspension
?. [Low Centrifugation]
AB1Material Supplier Info 215ml Falcon TubesDutsher (352097) 31.5 ml Eppendorf tubeEppendorf (0030 108.035)4RPMI 1640Thermofisher (11835030)
This step aims at clearing the suspension from debris and dead cells while preserving tumor cells. When working on cancerous tissues, we recommend not using Annexin V-based dead cell removal kits as they also remove altered but yet viable cells and presumed pre-apoptotic tumor cells that often represent relevant populations in the framework of single-cell studies. Successive low centrifugations were chosen instead as they allow to recover a higher proportion of tumor cells. This solution was also preferred over FACS sorting because of its significant time savings.
AB1Material Supplier Info 215ml Falcon TubesDutsher (352097) 31.5 ml Eppendorf tubeEppendorf (0030 108.035)4RPMI 1640Thermofisher (11835030)
?. [Low Centrifugation]
Transfer the cells in a 15ml Falcon tube and complete the suspension to a final volume of 12ml RPMI 1640.
?. [Low Centrifugation]
Centrifuge cell suspension at 800g
?. [Low Centrifugation]
Discard the supernatant and resuspend the pellet in 1 ml of RPMI 1640.
?. [Low Centrifugation]
Transfer cells in a new 15ml Falcon Tube and repeat the low centrifugation step.
?. [CD45 Positive Cells Depletion]
AB1Material Supplier Info 2CD45(TIL) MicroBeadsMiltenyi  Biotec (130-118-780)3EDTA 0.5 MSigma Aldrich (03690)4BSASigma Aldrich (A7906)5DPBSThermofisher (14190250)6RPMI 1640Thermofisher (11835030)7LS MACS ColumnsMiltenyi  Biotec (130-042-401)8MACS SeparatorMiltenyi  Biotec (130-090-976)
- This step is optional and depends on the targeted cell population. It aims at enriching the cell suspension with tumor and stromal cells by depleting the population of leucocytes, mainly consisting of circulating lymphocytes, which may represent the vast majority of the cells after dissociation.- The following volumes were adapted to a maximum population of 107 cells. In case you recover too much cells, please adapt the volumes according to the following ratios.
AB1Material Supplier Info 2CD45(TIL) MicroBeadsMiltenyi  Biotec (130-118-780)3EDTA 0.5 MSigma Aldrich (03690)4BSASigma Aldrich (A7906)5DPBSThermofisher (14190250)6RPMI 1640Thermofisher (11835030)7LS MACS ColumnsMiltenyi  Biotec (130-042-401)8MACS SeparatorMiltenyi  Biotec (130-090-976)
?. [CD45 Positive Cells Depletion]
Resuspend cell pellet (up to 107 cells) in 80 μl of MACS Buffer, and mix well by gently pipetting 10 to 15 times.
?. [CD45 Positive Cells Depletion]
Add 20 μl CD45 micro beads, mix and incubate in fridge
4 °C
?. [CD45 Positive Cells Depletion]
Add 400 µl of MACS Buffer, mix by pipetting
?. [CD45 Positive Cells Depletion]
Place LS column in QuadroMACS Magnetic Cell Separator and prepare it by rinsing with 3ml MACS Buffer (discard effluent).
?. [CD45 Positive Cells Depletion]
Apply cell suspension onto the column and immediately collect flow-through containing unlabeled cells in 1.5ml Falcon tube.
?. [CD45 Positive Cells Depletion]
Rinse the column twice with 1ml of MACS Buffer, collect and pool the effluent.
?. [CD45 Positive Cells Depletion]
Centrifuge cell suspension
?. [CD45 Positive Cells Depletion]
Discard the supernatant and resuspend the pellet in 1.5ml tubes with 250µl RPMI 1640.
Optionnal:- take 20µl from cell suspension for counting and microscopic quality control do the dissociation using trypan blue in a Malassez Chamber.- 0.25 million cells were used for Flow Cytometry Assays to measure viability with 7‐amino‐actinomycin (7AAD), as well as CD45 (immune) and CD34 (endothelial) positive cells.
?. [Tumor Preparation]
Place each biopsy onto 100mm Petri Dish, on ice
Depending on the amount of tissue, put aside parts of each biopsy for further complementary analyses.
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							- Number and identify each punch biopsy as well as its following match samples.
- Cut each biopsy in three parts (¼, ¼, ½) consisting of approximately ¼ for formaldehyde treatment and immunohistochemistry staining; ¼ for snap freezing, -80°C storage and further DNA/RNA extraction; and ½ for tissue dissociation of tumor punches.





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?. [Red Blood Cell (RBC) Lysis  Protocol]
Take advantage of the centrifugation duration to prepare 1X RBC Lysis Buffer on ice: dilute 1ml 10X RBC Lysis Buffer to 1X working concentration with 9ml deionized water.
?. [Red Blood Cell (RBC) Lysis  Protocol]
Discard the supernatant, gentlgy resuspend the cell pellet in 1ml RBC Lysis Buffer and quickly transfer it into a new 50ml Falcon.
?. [Red Blood Cell (RBC) Lysis  Protocol]
Add the remaining RBC Lysis Buffer.
?. [Red Blood Cell (RBC) Lysis  Protocol]
Incubate on Ice for 4 min with occasional shaking.
?. [Red Blood Cell (RBC) Lysis  Protocol]
Centrifuge cell suspension
Centrifuge: 300 34
?. [Red Blood Cell (RBC) Lysis  Protocol]
Discard the supernatant and resuspend the pellet in 1ml RPMI 1640
Optional:- Take 20µl from cell suspension for counting and microscopic quality control of the dissociation using trypan blue in a Malassez Chamber.- 0.3 million cells were used for Flow Cytometry Assays to measure viability with 7‐amino‐actinomycin (7AAD), as well as CD45 (immune) and CD34 (endothelial) positive cells..
?. [Red Blood Cell (RBC) Lysis  Protocol]
AB1  Material     Supplier Info  2  RBC Buffer (10x)    BioLegend (420301)  3  Desionized
  water    Invitrogen
  (10977035)  4  DPBS    Thermofisher
  (14190250)
In this step, red blood cells are lysed and washed away as they represent irrelevant cells for the study.
AB1  Material     Supplier Info  2  RBC Buffer (10x)    BioLegend (420301)  3  Desionized
  water    Invitrogen
  (10977035)  4  DPBS    Thermofisher
  (14190250)
?. [Tumor Dissociation]
Rinse the remaining cells in the MACS C tube and reapply it through the strainer.
?. [Tumor Dissociation]
Wash the strainer and complete the suspension to a final volume of 40ml RPMI 1640.
?. [Red Blood Cell (RBC) Lysis  Protocol]
Stop the reaction by diluting the active RBC Lysis Buffer with 30ml 1X PBS.
?. [CD45 Positive Cells Depletion]
Prepare 10ml MACS Buffer solution containing PBS, 0.5% BSA and 2 mM EDTA.
?. [Expected Result]
?. [Application]
Freezing samples and biobanking
- This step aims at storing the tumor cells after dissociation in order to allow their delayed study. This can be useful for the study of asynchronous samples such as matched primary and metastatic tumors.- We tested this approach and were able to obtain high quality and comparable data in single-cell RNA-sequencing of fresh versus frozen samples (data not shown).- Transfer enough cells (0.1 to 1 Million) in 1ml freezing solution consisting of 700µl RPMI 1640, 200µl Fetal Bovine Serum and 100µl DMSO). Immediately place the cells at -20°C for one hour before gradually freezing and storing them at -80°C overnight (~12h). Then, transfer cells to liquid nitrogen for long-term storage.
?. [Application]
Single Cell experiment
If cell viability is higher than 70%, cells can then be processed for scRNA-seq on 10X genomics platform. Gently resuspend the cells in appropriate volume of PBS, 0.04% BSA and proceed to 10X protocol.
?. [Tumor Preparation]
Material  AB1  Material     Supplier Info  2  Petri
  Dish, 100 mm    Corning
  (430591)  3  Biopsy
  Punch 8mm    Dutscher
  (030740)  4  Ice
This step should be designed according to the biological question. As this protocol was developed to catch the tumor heterogeneity, each tumor was sampled in multiple areas.
AB1  Material     Supplier Info  2  Petri
  Dish, 100 mm    Corning
  (430591)  3  Biopsy
  Punch 8mm    Dutscher
  (030740)  4  Ice
?. [Tumor Dissociation]
Prepare Digest Mix ON ICE by adding into a gentleMACS C Tube 4.7ml RPMI 1640, 200µl Enzyme H, 100µl Enzyme R and 25µl Enzyme A.
?. [Tumor Dissociation]
Gently mince the punch biopsies into small pieces of about 2 mm3 using a scalpel.
?. [Tumor Dissociation]
Using forceps, gently transfer tissue fragments into the gentle MACS C tube containing the Digest Mix (be careful not to crush the fragments). Add the remaining digest mix solution from the petri dish and tightly close C Tube.