Oct 26, 2023
Sputum Sample Processing for Single Cell Isolation and Live Recovery for Single Cell RNA Sequencing
- Jason D Limberis1,
- Alina Nalyvayko1,
- Michele Tomasicchio2,
- Shameem Jaumdally2,
- anil pooran2,
- Keertan Dheda2,
- john.metcalfe1
- 1University of California, San Francisco;
- 2University of Cape Town, South Africa
Protocol Citation: Jason D Limberis, Alina Nalyvayko, Michele Tomasicchio, Shameem Jaumdally, anil pooran, Keertan Dheda, john.metcalfe 2023. Sputum Sample Processing for Single Cell Isolation and Live Recovery for Single Cell RNA Sequencing. protocols.io https://dx.doi.org/10.17504/protocols.io.j8nlko9k5v5r/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: September 11, 2023
Last Modified: October 26, 2023
Protocol Integer ID: 87649
Keywords: sputum, single cell isolation, single cell RNA sequecncing, scRNAseq, infectious disease
Funders Acknowledgement:
UCB Catalyst
Grant ID: Chipotle
Abstract
Processing of clinical sputum samples for single live cell isolation for downstream applications.
Materials
Zymo DNA/RNA ShieldFisher ScientificCatalog #50-125-1706
Phosphate buffer solution (PBS)Contributed by users
EDTA (0.5 M), pH 8.0Life TechnologiesCatalog #AM9260G
Bovine Serum Albumin (BSA) Merck MilliporeSigma (Sigma-Aldrich)Catalog #A7906
Magnesium ChlorideFisher ScientificCatalog #AC223210010
DMSOMerck MilliporeSigma (Sigma-Aldrich)Catalog #472301
Fetal calf serum (FCS)Gemini Bio-ProductsCatalog # 900-108 DNase I, Bovine Pancreas, >500 Kunitz U/mgGold BiotechnologyCatalog #D-300
100 g N-Acetyl-L-CysteinebiorbytCatalog #orb320385
Collagenase, Type IV, powderThermo FisherCatalog #17104019
Before start
Increasing the amount of sputum, by asking the patient to expectorate several time, will likely increase the final cell numbers.
Prepare Buffers
Prepare Buffers
5mM EDTA in PBS
500ul 0.5M EDTA stock
49.5ml PBS. Store at 4C
10% BSA stock
Add 1g BSA to 10ml PBS, mix gently till dissolved
Filter sterilize & keep at 4C
50mM MgCl2 solution
Add 238mg of MgCl2 to 50ml of H2O
10% DMSO/FCS freezing mixture
5 mL DMSO
45 mL fetal calf serum (FCS)
7.5 Mass Percent NALC in PBS
DNase
Aliquoted into 100ul aliquots at 224kuml and stored at -20C
Add 100ul to 40ml dissociation buffer to make a final conc of 0.56ku/ml.
Collagenase IV
Add 500 µL to 40 mL dissociation buffer to make a final conc of 0.05mg/ml
Aliquoted 500 µL at 4mg/ml and stored at -20C.
Make fresh for each use
1% BSA in PBS
5 mL 10% BSA
45 mL PBS
Dissociation buffer
1.6 mL of 50 millimolar (mM) MgCl2
100 µL DNase
500 µL collagenase
37.8 mL PBS
Sputum collection
Sputum collection
Collect an induced sputum, preferably 5 mL or more in volume, and process as below as soon as possible.
Weigh the sputum collection cup before and after the sputum collection.
Estimate the volume of sputum collected using a graduated container of the same size.
Record the viscosity (salivary, mucosalivary, purulent, mucopurulent)
Record the time of sputum collection
Bulk RNA sequencing
Bulk RNA sequencing
Record the time of sputum processing
Using a wide bore tip (or cut the bottom of a 1000 µL ) draw up ~200 µL sputum. If the sample is too viscous for a pipette, use a syringe needle instead.
Add to a 1.5ml tube containing 500 µL Zymo RNA Shield
Vortex tube for 00:00:10
10s
Immediately transfer to -80 °C
Make a single cell suspension (scRNAseq)
Make a single cell suspension (scRNAseq)
Add 40 mL dissociation buffer into the collection tube containing the sputum sample
Secure cap, manually invert the tube several times. Secure to a hula mixer (low setting) and incubate for 00:10:00 at Room temperature .
Note: Big clumps should dissolve after 00:10:00 . Some small fibers may will remain.
20m
Add DTT to a final concentration of 1-2mM.
Invert the tube several times and incubate for 00:05:00
5m
Pass the dissociated sample through a 100 µm strainer on top of a new 50 mL tube using an automated pipettor (do not pour). 350 rcf, 4°C, 00:10:00
10m
Wash the strainer with 2 mL of PBS
Spin tube in a sealed bucket at 250 rcf, 4°C, 00:10:00
10m
Remove 1000 µL supernatant and place in a cryovial for Luminex Cytokine assays. Add 140 µL of protease inhibitor and store at -80 °C
Aspirate the remainder of the supernatant and discard.
Gently resuspend cell pellet in 1 mL EDTA/PBS using a wide bore 1000 µL tip
Count cells using Turks protocol followed by paraformaldehyde fixation to decontaminate
Long-term storage of cells (scRNAseq)
Long-term storage of cells (scRNAseq)
Pre-cool CellCool and cryovials at 4 °C for >02:00:00
2h
Place cells On ice
Gently mix the cells
Centrifuge at 300 rcf, 4°C, 00:05:00
5m
Discarded supernatant and resuspend cell pellets in pre-chilled (4 °C ) 10% DMSO/FCS solution (add dropwise)
Dispense cell suspension in 500 µL aliquots into the pre-cooled cryovials and place into CoolCell at -80 °C
Expected results
Expected results
Pilot samples
| A | B | C | D | |
| Sample | S1 | S2 | S3 | |
| Viscosity | mucosalivary | mucosalivary | mucosalivary | |
| Volume used (ml) | 10 | 4 | 6 | |
| Total cells before freezing (10^6) | 2.2 | 0.9 | 2.9 | |
| Total cells recovered after thawing (2 weeks after frozen; 10^6) | 0.88 | 0.48 | 1.33 | |
| Percentage cell recovery post freezing (%) | 40 | 53 | 46 |
S2 400x
S3 100x
Clinical samples
| A | B | C | D | E | F | G | H | I | J | K | L | M | N | |
| Sample ID | Time to sputum processing (h.m) | weight of sputum (g) | volume of sputum (ml) | viscosity of sputum | Time to thaw (days) | Total cell count pre freeze (x10^6) | Total cell count taken forward pre freeze (x10^6) | Total cell count post freeze (x10^6) | Total cell count difference (x10^6) | Live cell count | Dead cell count | Viability (%) | Viability of prefreeze total (%) | |
| CC20 | 2.85 | 17.8 | 15 | salivary | 662 | 9.1 | 7 | 4.86 | 2.14 | 1.58 | 3.28 | 33 | 23 | |
| CC22 | <6.00 | 2.1 | 2 | mucoid | 658 | 1.3 | 1.3 | 0.60 | 0.71 | 0.15 | 0.44 | 26 | 12 | |
| CC23 | 2.10 | 2.9 | 3 | mucosalivary | 657 | 0.43 | 0.43 | 0.37 | 0.06 | 0.08 | 0.29 | 21 | 18 | |
| CC24 | 1.10 | 8.7 | 8 | mucosalivary | 657 | 1.1 | 1.1 | 0.81 | 0.30 | 0.19 | 0.62 | 23 | 17 | |
| CC25 | 4.90 | 0.7 | 0.9 | mucosalivary | 652 | 0.17 | 0.17 | 0.00 | 0.17 | 0.00 | 0.00 | 0 | 0 | |
| CC26 | 2.00 | 1.5 | 1.4 | mucosalivary | 652 | 1.13 | 1.13 | 0.73 | 0.40 | 0.17 | 0.57 | 23 | 15 | |
| CC27 | 2.20 | 3.2 | 3 | mucosalivary | 651 | 1.4 | 1.4 | 1.33 | 0.07 | 0.30 | 1.03 | 23 | 22 | |
| CC28 | 2.00 | 12 | 10 | mucoid | 644 | 3.2 | 2 | 2.74 | -0.74 | 0.29 | 2.45 | 11 | 15 | |
| CC33 | 2.00 | 3 | 3 | mucosalivary | 627 | 0.8 | 0.8 | 0.67 | 0.13 | 0.25 | 0.42 | 37 | 31 | |
| CC37 | <6.00 | 4.1 | 4 | mucosalivary | 623 | 0.92 | 0.92 | 0.97 | -0.05 | 0.34 | 0.63 | 35 | 37 |