Bacterial Single-Cell RNA-seq Protocol

Single Cell Isolation and Lysis

1. Streak Salmonella and Pseudomonas wild type on LBA and Salmonella GFP-tagged strain on LBA plates containing Chloramphenicol. Grow them overnight at 37 °C.
2. Start overnight cultures: Pick a single colony from agar plate and inoculate 10 ml glass tubes filled with 2 ml LB media (supplemented with 20 μg/ml Chloramphenicol for GFP strain).
3. Prepare 50 ml Erlenmeyer flasks with 20 ml LB. Add 200 μl of overnight culture (OD 1) – starting OD 0.01
4. Incubate at 37 °C with agitation.
5. Shock conditions: Grow until OD 0.3, then add NaCl to a final concentration of 0.3 M and incubate for 10 min at 37°C with agitation (for NaCl shock), respectively, add a layer of mineral oil and incubate for 30 min at 37 C without agitation.
6. Late stationary phase: Grow until OD 2.0, followed by further 6 hours growth at 37 C with agitation.
7. Prepare lysis mix meanwhile cultures grow.
8. Lysis mix for 20 reactions: 39 μl ddH₂O, 5.2 μl 10 × lysis buffer, 5.2 μl PBS 10 ×, 2 μl Lysozyme (50 U/μl) and 0.6 μl RNase Inhibitor. Vortex briefly and spin down.
9. For Pseudomonas: Add EDTA to a final concentration of 0.5 mM and DTT to a final concentration of 100 mM. Adjust volume of ddH₂O in previous step accordingly.
10. Use either 48 or 96-well plate and add 2.6 μl of lysis buffer per well. Seal plates properly and store at 4 °C until sorting.
11. As soon as cultures are ready, take 1 ml of each culture and spin down immediately for 4 min at 17,000 × g, washing procedure is done at room temperature.
12. Take off supernatant carefully without touching pellet.
13. Resuspend pellet in 1 ml PBS.
14. Spin down immediately for 4 min at 17,000 × g, room temperature.
15. Repeat washing procedure (steps 13 and 14) once again.
16. Prepare 1:1 1 PBS, RNAlater mix (1 ml per sample).
17. Resuspend pellet in PBS, RNAlater mix.
18. Store samples on ice until sorting.
19. Start FACS and cooling system. Perform fluidics start-up.
20. Insert 70 nozzle and perform Accudrop delay after stream is stable.
21. Adjust automated cell deposition unit (ACDU) adaptor and home device settings to 96-well plate format.
22. Dilute samples in 1 PBS, at least 1:10.
23. Load GPF-tagged strain.
24. Increase the flow rate to max for about 30 s to ensure stable detection of events.
25. Lower flow rate to achieve about 1000–1500 events/s.
26. Check events on Forward/ Sideward Scatter (SSC/ FSC) plot and check counts for FITC-positive cells as well.
27. FITC-positive cells are used as marker to identify bacterial cells.
28. Perform selection of fluorescein isothiocyanate (FITC-) positive cells and apply selection on SSC/ FSC plot to identify events resulting from GFP-tagged bacteria.
29. The sorting gate on SSC/ FSC plot is defined as population 1 and contains FITC-positive cells only.
30. Save gating strategy as global worksheet.
31. Unload GFP-tagged strain and load WT strains.
32. Increase the flow rate to max for about 30 s to ensure stable detection of events.
33. Lower flow rate to achieve about 1000–1500 events/s.
34. Create sort scheme: Use single-cell precision. Include at least one negative (NC) and one positive control (PC) on each sorted plate. Exclude those wells from sorting.
35. Spin down plate containing lysis buffer and put on 96-well plate adapter on FACS.
36. Sort cells with about 1000–1500 events/s.
37. Seal plate properly using Microseal "F", spin down quickly, keep on ice, and store at -80 °C for up to 6 months.
38. For Pseudomonas: Sonicate plate for 10 s after spin down, keep on ice, and store afterwards at 80 °C.

Reverse Transcription

1. Prepare the primer mix by adding 7.08 μl ddH₂O, 0.12 μl GAT27 dT primer, each 0.4 μl of GAT27 5N3G and GAT27 5N3T into 0.5 ml tube. Vortex and spin down.
2. Mix 0.4 μl of primer mix with 0.05 μl DTT and 0.12 μl dNTP. Vortex and spin down pre-RT mix.
3. Positive control: Add 0.5 μl of DNase treated total RNA (100 pg/μl, total input: 50 pg).
4. Negative control: Add 0.5 μl ddH₂O.
5. Add 0.57 μl of pre-RT mix to each well containing sorted cell in lysis buffer. Vortex and spin down.
6. Incubate for 3 min at 72 °C, transfer on ice for at least 1 min.
7. Prepare RT mix: 1.15 μl ddH₂O, 0.8 μl Superscript III buffer, 0.2 μl DTT, 0.1 μl RNase Inhibitor, and 0.15 μl Superscript III.
8. Add 2.4 μl of RT mix to each well. Vortex and spin down.
9. Run PCR. Proceed immediately with Primer Digestion.

Primer and RNA Digestion

1. Incubate 1 min at 50 °C.
2. Perform this step at room temperature: Add 0.2 μl T4 DNA Polymerase. Vortex and spin down.
3. Run PCR.
4. Put plate on ice or cooling block. Add 0.1 μl of RNase H and RNase If. Vortex and spin down.
5. Run PCR.

Tailing

1. Add 3.13 μl ddH₂O, 0.4 μl dCTP, 0.4 μl TdT-buffer, and 0.1 μl ddH₂O Terminal Transferase. Vortex and spin down.
2. Run PCR.

Second-Strand Synthesis

1. Add 12.925 μl ddH₂O, 1.5 μl Thermopol buffer, 1.25 μl dNTP, and 0.125 μl GAT21 6NG3 primer. Vortex and spin down.
2. Incubate 1 min at 95 °C, hold at 48 °C.
3. Perform this step at room temperature: Add 0.4 μl Deep Vent (exo-) DNA Polymerase. Vortex and spin down.
4. Run PCR.

PCR Amplification

1. Add 114 μl ddH₂O, 13 μl Thermopol buffer, 3 μl dNTP, 3 μl Deep Vent (exo-) DNA polymerase, and 0.8 μl GAT27 PCR primer. Mix 10 times up and down
2. Distribute each reaction into 4 wells (40 μl each). Vortex and spin down.
3. Run PCR.
4. Safe stopping point: Plate can be stored at -20 °C for up to two days. Otherwise continue directly with cleanup procedure.

Cleanup with Magnetic Beads

1. Bring AMPure XP beads at room temperature at least 30 min in advance.
2. Take out sample plate and keep at room temperature for 15 min. Spin down briefly before continuing.
3. Vortex beads vigorously and make sure beads are well suspended.
4. Add 40 μl of beads to every well and mix up and down several times.
5. Incubate for 10 min.
6. Place the plate onto a 96-well magnet and incubate for 2 min or until the supernatant is completely clear.
7. Remove the supernatant carefully without touching the pellet.
8. Add 180 μl of fresh 80% ethanol to each well and incubate for 30 s on the magnetic stand.
9. Remove and discard ethanol without removing the PCR plate from the magnet.
10. Repeat washing procedure (steps 8, 9) once again.
11. Remove residual ethanol with 10 or 20 μl pipette. Make sure no ethanol is remaining in wells. Keep plate on magnet.
12. Air-dry on magnet for about 8 min. Carefully check when the beads are dry. The best indicator is lack of shininess and appearing cracks.
13. Remove plate from magnet.
14. Add 13.1 μl ddH₂O to each well and mix by pipetting up and down until all beads are eluted.
15. Incubate for 2 min at room temperature.
16. Place plate back on magnet and incubate for 2 min or until the supernatant is completely clear.
17. Transfer 11 μl to a new 48-/96-well plate.
18. Safe-stopping point: Plate can be stored at -20 °C for up to 6 months.

Quality Control of cDNA

1. Perform Qubit dsDNA High Sensitivity assay for every sample and check DNA concentration.
2. Dilute samples to 1 ng/μl and run them on Bioanalyzer DNA HS chip.

Library Preparation and Pooling

1. Sample input: 1.25 μl cDNA (normalized to 0.4 ng/μl, total of 0.5 ng).
2. Only 1/4 of reaction/ indices volume is used.
3. Tagmentation time: 10 min.
4. Quality control of libraries: follow QC of cDNA.
5. Dilute libraries to 5 nM in ddH₂O and pool samples equimolar.
6. Quality control of pool: follow QC of cDNA.

Sequencing and Demultiplexing

1. Sequencing is performed in paired-end mode.
2. Sequencing depth: at least 20 M reads per single cell.
3. Demultiplexing of raw sequencing data is performed.
4. Data analysis after demultiplexing includes quality control (FastQC), adaptor trimming (cutadapt), and mapping (STAR aligner). Differential gene expression analysis is performed using DESeq2 package and read counts for each gene are determined using featureCounts algorithm. Thereby about 93% of mapped reads account for ribosomal as well as transfer RNA.