Oct 26, 2023

Sputum Sample Processing for Single Cell Isolation and Live Recovery for Single Cell RNA Sequencing

DOI
https://dx.doi.org/10.17504/protocols.io.j8nlko9k5v5r/v1
  • 1University of California, San Francisco;
  • 2University of Cape Town, South Africa
Jason D Limberis
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DOI: https://dx.doi.org/10.17504/protocols.io.j8nlko9k5v5r/v1
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, clinical sputum samples for single live cell isolation, live recovery for single cell rna sequencing processing, single cell rna sequencing processing, single live cell isolation, clinical sputum sample, single cell, rna, cell, live recovery
Funders Acknowledgements:
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)
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
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
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
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)
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)
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
Pilot samples
ABCD
SampleS1S2S3
Viscositymucosalivary mucosalivary mucosalivary
Volume used (ml)1046
Total cells before freezing (10^6)2.20.92.9
Total cells recovered after thawing (2 weeks after frozen; 10^6)0.880.481.33
Percentage cell recovery post freezing (%)405346


S2 400x
S3 100x
Clinical samples
ABCDEFGHIJKLMN
Sample IDTime to sputum processing (h.m)weight of sputum (g)volume of sputum (ml)viscosity of sputumTime 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 countDead cell countViability (%)Viability of prefreeze total (%)
CC202.8517.815salivary6629.174.862.141.583.283323
CC22<6.002.12mucoid6581.31.30.600.710.150.442612
CC232.102.93mucosalivary6570.430.430.370.060.080.292118
CC241.108.78mucosalivary6571.11.10.810.300.190.622317
CC254.900.70.9mucosalivary6520.170.170.000.170.000.0000
CC262.001.51.4mucosalivary6521.131.130.730.400.170.572315
CC272.203.23mucosalivary6511.41.41.330.070.301.032322
CC282.001210mucoid6443.222.74-0.740.292.451115
CC332.0033mucosalivary6270.80.80.670.130.250.423731
CC37<6.004.14mucosalivary6230.920.920.97-0.050.340.633537