TITLE: Sputum Sample Processing for Single Cell Isolation and Live Recovery for Single Cell RNA Sequencing
AUTHORS: Jason D Limberis, Alina Nalyvayko, Michele Tomasicchio, Shameem Jaumdally, anil pooran, Keertan Dheda, john.metcalfe

[DESCRIPTION]
Processing of clinical sputum samples for single live cell isolation for downstream applications.

[BEFORE_START]
Increasing the amount of sputum, by asking the patient to expectorate several time, will likely increase the final cell numbers.

[STEPS]
SECTION: Sputum collection
2. Collect an induced sputum, preferably 5 mL or more in volume, and process as below as soon as possible.

SECTION: Bulk RNA sequencing
4. 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.

SECTION: Bulk RNA sequencing
5. Add to a 1.5ml tube containing 500 µL Zymo RNA Shield

SECTION: Bulk RNA sequencing
6. Vortex tube for 10 s

SECTION: Bulk RNA sequencing
7. Immediately transfer to -80 °C 
SECTION: Make a single cell suspension (scRNAseq)
8. Add 40 mL dissociation buffer into the collection tube containing the sputum sample

SECTION: Make a single cell suspension (scRNAseq)
9. Secure cap, manually invert the tube several times. Secure to a hula mixer (low setting) and incubate for 10 min at Room temperature. 

Note: Big clumps should dissolve after 10 min. Some small fibers may will remain.

SECTION: Make a single cell suspension (scRNAseq)
10. Add DTT to a final concentration of 1-2mM. 
Invert the tube several times and incubate for 5 min

SECTION: Make a single cell suspension (scRNAseq)
11. Pass the dissociated sample through a   strainer on top of a new 50 mL tube using an automated pipettor (do not pour). 350 rcf, 10 min, 4 °C

SECTION: Make a single cell suspension (scRNAseq)
12. Wash the strainer with 2 mL of PBS

SECTION: Make a single cell suspension (scRNAseq)
13. Spin tube in a sealed bucket at 250 rcf, 10 min, 4 °C

SECTION: Make a single cell suspension (scRNAseq)
14. Remove 1000 µL supernatant and place in a cryovial for Luminex Cytokine assays. Add 140 µL of protease inhibitor and store at -80 °C

SECTION: Make a single cell suspension (scRNAseq)
15. Aspirate the remainder of the supernatant and discard.
SECTION: Make a single cell suspension (scRNAseq)
16. Gently resuspend cell pellet in 1 mL EDTA/PBS using a wide bore 1000 µL tip

SECTION: Make a single cell suspension (scRNAseq)
17. Count cells using Turks protocol followed by paraformaldehyde fixation to decontaminate
SECTION: Long-term storage of cells (scRNAseq)
18. Pre-cool CellCool and cryovials at 4 °Cfor >120 min

SECTION: Long-term storage of cells (scRNAseq)
19. Place cells on ice

SECTION: Long-term storage of cells (scRNAseq)
20. Gently mix the cells
SECTION: Long-term storage of cells (scRNAseq)
21. Centrifuge at 300 rcf, 5 min, 4 °C

SECTION: Long-term storage of cells (scRNAseq)
22. Discarded supernatant and resuspend cell pellets in pre-chilled (4 °C) 10% DMSO/FCS solution (add dropwise)

SECTION: Long-term storage of cells (scRNAseq)
23. Dispense cell suspension in 500 µL aliquots into the pre-cooled cryovials and place into CoolCell at -80 °C

SECTION: Expected results
24. Pilot samples 
     Sample      S1      S2      S3        Viscosity      mucosalivary      mucosalivary      mucosalivary        Total
  volume (ml)      21      8      12        Weight
  (g)      22.6      7      11.8        volume       10
  ml      4ml      6ml        Total
  cells before freezing (cells stored at 5x10^6/ml at -80C)      2.2x10^6       0.9x10^6      2.9x10^6        Total
  cells recovered after thawing (2 weeks after frozen)      0.88x10^6      0.48x10^6      1.33x10^6        Percentage
  cell recovery post freezing      40%      53%      46%        Microscope
  post thaw        400x               100x   

  

       
 
 Clinical samples
      CHIPOLTLE
  sample ID (written on tube)  Time to
  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  Unknown  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  Unknown  4.1  4  mucosalivary  623  0.92  0.92  0.97  -0.05  0.34  0.63  35%  37%  

SECTION: Sputum collection
2.1. Weigh the sputum collection cup before and after the sputum collection.
SECTION: Sputum collection
2.2. Estimate the volume of sputum collected using a graduated container of the same size.
SECTION: Sputum collection
2.3. Record the viscosity (salivary, mucosalivary, purulent, mucopurulent)
SECTION: Sputum collection
2.4. Record the time of sputum collection
SECTION: Bulk RNA sequencing
3. Record the time of sputum processing
SECTION: Prepare Buffers
1. 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 mLDMSO
    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