TITLE: Processing of human surgical samples for single-cell sequencing
AUTHORS: Álvaro Quintanal-Villalonga,    Viola Allaj,    Andrew Chow,    Nisarg Shah,    Linas Mazutis, John  Thomas Poirier, Triparna Sen, Dana Pe'er, Charles  M. Rudin

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Human clinical samples pose a challenge for single-cell RNA-seq (scRNA-seq) and single-nucleus ATAC-seq (snATAC-seq) analysis, due to their heterogeneity in cell type composition and degree of degradation or necrosis. Moreover, although some tissues can be sampled by surgical resection, most biospecimens are derived from core needle biopsies or fine needle aspirates, and must be allocated to pressing needs such as pathological analysis, thereby severely limiting the number of cells available for analysis. We have developed a protocol for obtaining robust, high-quality single-cell sequencing data from human biospecimens from a variety of biopsy types, enabling greater access to clinical cohorts. A critical factor is to limit ischemic time and transport to the lab to under an hour, and to restrict subsequent processing time to under 3 hours. This protocol uses a commercial tissue disruptor for dissociation. It was optimized on diverse human lung tumor samples, including pleural effusions, but is widely applicable to normal and diseased human clinical tissues from different organ sites.</div></div>

[STEPS]
?. [Sample collection and transport to the lab]
Rapid transportation of the surgical sample to the lab after resection or biopsy is key to the success of this protocol. To maximize cell viability and sequencing data quality, processing of clinical samples in the lab should begin within an hour of surgical collection, and never more than two hours after.
?. [Sample collection and transport to the lab]
Surgical samples should be placed in PBS, saline (0.9% w/v sodium chloride) or RPMI media after resection, and transported to the lab on ice.
?. [Sample staining]
Staining will allow dead cells to be selectively removed based on calcein AM and DAPI stains. Additionally, CD45 staining will allow live cells to be further sorted by CD45 marker status to separate immune (CD45+) from non-immune, tumor-enriched (CD45-) cell fractions.
The goal of CD45 staining and sorting is to enrich tumor cells in samples for which these make up a relatively small fraction of the total tumor mass (e.g. lung adenocarcinoma). CD45 staining does not need to be performed if tumor cell enrichment is not required; however, sorting for live (DAPI-, Calcein+) cells is always required to fulfill the high viability requirements of library preparation and sequencing steps.
?. [Sample staining]
Centrifuge the FACS tube  and aspire supernatant carefully so as not to disturb the pellet.
Centrifuge: 800 34, 3 min
?. [Sample staining]
Add  of staining cocktail per ~1 million cells and vortex briefly to mix.
100 µl
Staining cocktail preparation (per  ) for live and CD45+/- cell sorting:-   PBS 2.5% FBS-   of human TruStain FcX-   of PE anti-human CD45 antibody-   of   calcein AMStaining cocktail preparation (per  ) for live cell sorting:-   PBS 2.5% FBS-   of   calcein AM
?. [Sample staining]
Incubate cells with staining cocktail for  on ice or at  .
4 °C
?. [Sample staining]
Wash by adding  of PBS 2.5% FBS and centrifuging at . Aspirate supernatant carefully and repeat once, for a total of two washes.
2 mL
Centrifuge: 800 34, 3 min
?. [Sample sorting]
Cell sorting can be performed on any standard FACS instrument.
?. [Sample sorting]
Resuspend cells in  RPMI 2.5% FBS per million cells with DAPI (final concentration of 0.1-1 μg/mL), and place on ice.
100 µl
?. [Sample sorting]
Set sorting gates to capture the cell populations of interest. If tumor cell enrichment is required and was performed in Step 3, set the following gates (example below) and record cell fractions in your notes:- Live immune cells: DAPI-, FITC+ (Calcein+), PE+- Live non-immune (tumor-enriched) cells: DAPI-, FITC+ (Calcein+), PE-Alternatively, if tumor cell enrichment is not required and CD45 staining was not performed, set the following gates:- Live cells: DAPI-, FITC+ (Calcein+)
For smaller samples, using more permissive (lower purity) sorting conditions improves cell recovery, ensuring enough cells for successful sequencing library preparation.
?. [Sample sorting]
If CD45 staining and sorting was performed, tumor cell enrichment can be performed by mixing immune (CD45+) and non-immune (tumor-enriched, CD45-) cell fractions back together at the desired ratio, according to your research interests.
?. [Sample sorting]
Prepare Eppendorf tubes containing  of RPMI 2.5% FBS that will serve as sort collection tubes.
50 µl
?. [Sample sorting]
Concentrate sample by centrifuging  in an oscillating rotor centrifuge, very carefully aspire supernatant, and resuspend in RPMI 2.5% FBS to 700 cells/µL.
Centrifuge: 800 34, 3 min, 4 10
The use of a swinging bucket centrifuge is ideal for maximizing cell recovery.
?. [Sample preparation]
The specimen first needs to be processed to obtain a single-cell suspension that is free of red blood cells. For typical resections (>100 mg), proceed to Section 3. For core needle biopsies (at least one core, typically 10-100 mg) or fine needle aspirates (typically 10-50 mg), proceed to Section 4. For pleural effusion samples, proceed to Section 5.
It is important to minimize the dissociation and processing time as much as possible, to improve cell viability and quality of sequencing data.
?. [Surgical specimen dissociation]
Resuspend cells in  to  of PBS 2.5% FBS and transfer to a Blue cap 35 µm filter FACS tube (Corning) by pipetting onto the cap filter. Proceed to Section 6.
3 mL
4 mL
?. [Surgical specimen dissociation]
Add  to  PBS 2.5% FBS to dilute the ACK buffer, centrifuge again at , and aspirate supernatant carefully so as not to disturb the pellet.
20 mL
25 mL
Centrifuge: 800 34, 3 min
At this point, the pellet should be whitish and not red. If it still looks red, repeat steps 3.7 and 3.8.
?. [Surgical specimen dissociation]
Gently resuspend cell pellet in  of Red Blood Cell Lysis Solution (ACK buffer) (Lonza) by pipetting up and down 2-3 times. Incubate for 2-3 minutes.
1 mL
RBC lysis should be included even if blood is not visually evident in the sample.
?. [Surgical specimen dissociation]
Spin  and aspire supernatant carefully so as not to disturb the pellet.
Centrifuge: 800 34, 3 min
?. [Surgical specimen dissociation]
Wash the 70 μm MACS SmartStrainer gently with  to  of PBS 2.5% FBS buffer. Remove strainer.
20 mL
25 mL
?. [Surgical specimen dissociation]
Apply the cell suspension to a 70 μm MACS SmartStrainer (Miltenyi) placed in a 50 mL conical tube.
?. [Surgical specimen dissociation]
Place the GentleMACS C Tube in the GentleMACS Octo Dissociator with Heaters (Miltenyi), and run program 37C_H_TDK3 for . After this time, check the solution to ensure complete dissociation (no tissue chunks visible to the naked eye). If the sample is not completely dissociated, continue running the program for rounds of  until completely dissociated. Do not continue any longer than necessary, as extended incubation can damage fragile cells and reduce RNA quality.
?. [Surgical specimen dissociation]
Aspirate the liquid in which the tumor has been submerged and transfer the tumor to a petri dish. Chop into ~5 mm3 pieces using a new sharp razor, and transfer the tumor pieces to the GentleMACS C Tube containing the enzyme mix.
?. [Surgical specimen dissociation]
Add  of Enzyme H,  of Enzyme R, and  μL of Enzyme A from HumanTumor Dissociation Kit (Miltenyi) into a GentleMACS C Tube (Miltenyi) with  of medium (RPMI without antibiotics or FBS).
250 µl
110 µl
40 µl
7.5 µl
?. [Surgical specimen dissociation]
This standard protocol should be used when tissue is not limiting.
?. [Small specimen processing]
Smaller samples are more effectively dissociated in reduced volumes for shorter periods, and extra care needs to be taken to avoid cell loss at each step.
?. [Pleural effusion processing]
Pleural effusions are cell suspensions that can be effectively concentrated from much larger volumes using density gradient centrifugation.
?. [Small specimen processing]
Add  of Enzyme H,  of Enzyme R, and  μL of Enzyme A from HumanTumor Dissociation Kit (Miltenyi) into a GentleMACS C Tube (Miltenyi) with  of medium (RPMI without antibiotics or FBS).
85 µl
40 µl
15 µl
2.5 µl
?. [Small specimen processing]
Aspirate the liquid in which the tumor has been submerged and transfer the tumor pieces to the GentleMACS C Tube containing the enzyme mix.
If the cores are disaggregated, centrifugation at   is recommended to collect all tumor pieces at the bottom of the tube before liquid aspiration and transferring to the GentleMACS C Tube containing the enzyme mix.
?. [Small specimen processing]
Place the GentleMACS C Tube in the GentleMACS Octo Dissociator with Heaters (Miltenyi), and run program 37C_H_TDK3 for . After this time, check the solution to ensure complete dissociation (no tissue chunks visible to the naked eye). If the sample is not completely dissociated, continue running the program for rounds of  until completely dissociated. Do not continue any longer than necessary, as extended incubation can damage fragile cells and reduce RNA quality.
?. [Small specimen processing]
Apply the cell suspension to a Falcon™ Test Tube with 35 µm Cell Strainer Snap Cap (Corning) though the cell strainer cap. To improve cell recovery, add  of PBS 2.5% FBS to the GentleMACS C Tube and wash the tube with it. Then, add it to the same Falcon™ Test Tube with 35 µm Cell Strainer Snap Cap (Corning) though the cell strainer cap.
1.5 mL
?. [Small specimen processing]
Spin  and aspire supernatant carefully so as not to disturb the pellet.
Centrifuge: 800 34, 2 min
?. [Small specimen processing]
Gently resuspend cell pellet in  of Red Blood Cell Lysis Solution (ACK buffer) (Lonza) by pipetting up and down 2-3 times. Incubate for 2-3 minutes.
0.2 mL
RBC lysis should be included even if blood is not visually evident in the sample.
?. [Small specimen processing]
Add  to  PBS 2.5% FBS to dilute the ACK buffer, centrifuge again at , and aspire supernatant carefully so as not to disturb the pellet. Proceed to Section 6.
2 mL
3 mL
Centrifuge: 800 34, 2 min
At this point, the pellet should be whitish and not red. If it still looks red, repeat steps 4.6 and 4.7.
?. [Pleural effusion processing]
To concentrate the cells, divide the fluid into conical tubes and centrifuge at . Carefully remove supernatant and resuspend the pellets into  to  of PBS 2.5% FBS.
Centrifuge: 500 34, 10 min
10 mL
40 mL
The resuspension volume depends on the size of the pellets obtained. Typically,   of PBS 2.5% FBS are used per   of initial fluid volume.
?. [Pleural effusion processing]
Prepare SepMate™-50 (IVD) tubes (STEMCELL Technologies) by pipetting ~ of Ficoll-Paque PLUS (GE Healthcare) directly and gently into tube’s separating hole until the fluid level is just above the dividing plastic. One  SepMate™-50 (IVD) tube will be needed per  of concentrated cell suspension in PBS 2.5% FBS .
15 mL
20 mL
?. [Pleural effusion processing]
Carefully layer  concentrated cell suspension in PBS 2.5% FBS onto the Ficoll-Paque PLUS in the  SepMate™-50 (IVD) tube (drop by drop).
20 mL
When layering the sample, try to minimize mixing of the Ficoll-Paque PLUS solution and the concentrated cell suspension.
?. [Pleural effusion processing]
Centrifuge at .
Centrifuge: 1200 34, 10 min
?. [Pleural effusion processing]
Pipette off  of upper fluid layer from each SepMate™-50 (IVD) tube into a Falcon tube. For the remaining ~ above the dividing plastic, try to pipette up and down gently to loosen and collect the residue sitting above the dividing plastic.
15 mL
5 mL
?. [Pleural effusion processing]
Centrifuge at  and discard supernatant.
Centrifuge: 800 34, 3 min
?. [Pleural effusion processing]
Gently resuspend cell pellet in  of Red Blood Cell Lysis Solution (ACK buffer) (Lonza) by pipetting up and down 2-3 times. Incubate for 2-3 minutes.
1 mL
RBC lysis should be included even if blood is not visually evident in the sample.
?. [Pleural effusion processing]
Add  to  PBS 2.5% FBS to dilute the ACK buffer, centrifuge again at , and aspire supernatant carefully so as not to disturb the pellet. Proceed to Section 6.
20 mL
25 mL
Centrifuge: 800 34, 3 min
At this point, the pellet should be whitish and not red. If it still looks red, repeat steps 5.7 and 5.8.