3.1. Oral rinse collection • TIMING: 1-1.5 hrs
CRITICAL: Maintain each step of primary saliva neutrophil isolation at room temperature (RT) as the cells tend to lose viability faster at a colder temperature (e.g. 4 ℃) that is generally used for PBMCs or any other primary cell type isolation.
1) Label the sterile 50 mL centrifuge tubes for collection of oral rinse and set the centrifuge to RT.
2) Before collecting the first oral rinse, each donor must clean their oral cavity and wait for 3 min.
3) Each donor must rinse their oral cavity five times with ~10 mL of 0.9 % NaCl solution (sterile) for 30-60 sec each time with a gap of 3 min between each rinse to collect 50 mL total volume. (CRITICAL: The collection tube should be immediately proceeded for the enrichment process, as neutrophil RNA keeps degrading over time.)
3.2. Saliva Neutrophil Isolation/enrichment • TIMING: 1-1.5 hrs
4) Pellet down the cells by centrifuging at 160 x g at RT for 5-10 min. (Alternatively, if there is high mucus content in the sample, the cell suspension can be passed through 40 µm sterile cell strainer before centrifugation).? TROUBLESHOOTING
5) Discard 40 mL of supernatant from 50 mL total vol. by aspirating carefully without disturbing the cell pellet. (CRITICAL: Neutrophils do not form a solid pellet)
6) Resuspended the cells in the remaining ~10 mL of 0.9 % saline solution and passed through 40 µm sterile cell strainer using gravity to remove any food particles or mucus present in the collected oral rinse.? TROUBLESHOOTING
7) Then sequentially filter the 40 µm filtered oral rinse through pluriStrainer nylon mesh filters 20 µm and 10 µm to remove epithelial cells. (CRITICAL: All filtration steps are to be done by gravity flow or centrifuge at 160 x g at RT for 1 min. Avoid using vacuum suction as this suck’s in smaller epithelial cells through the 20 µm & 10 µm pluriStrainer. Check under an optical microscope after each filtration process to ensure removal of larger cell and food particles. If necessary, filter again. Check cell viability and density after each filtration step. The cell viability tends to increase with reduction of epithelial cells, which are mostly dead and add to reduced cell viability before 20 and 10 µm filtration).
8) Count the cells to check the viability and cell density by staining 10 µL aliquot of filtered cell suspension with trypan blue (10 µL) and loading into Countess cell counting chamber slides and using Countess automated cell counter. (Expected total number of cells obtained from a healthy donor in 50 mL oral rinse of resting saliva is ~1.8 x 10^5 cells having viability of 70-80 %. This procedure provides > 95 % enriched saliva neutrophils). (CRITICAL: Proceed immediately to the downstream experimental step requiring unfixed cells. Otherwise, for downstream experiments requiring fixed cells, proceed with cell fixation by 4 % PFA).? TROUBLESHOOTING
3.3. FACS sorting • TIMING: 2-3 hrs
(CRITICAL: The FACS equipment for sorting should be prepared and kept ready beforehand to minimize the exposure of saliva neutrophil in its non-native environment.) [NOTE: All provided timing is for single 384-well plate]
On verifying the desired purity (i.e. > 95 %) by observing under optical microscope (Fig. 2) and determining > 70 % cell viability by trypan blue exclusion method (using Countess cell counter) of enriched neutrophils (Supplementary Fig. 5), proceed immediately with FACS sorting. As we performed unbiased cell sorting, we did not stain the cells with any antibody/marker and used the cell suspension in saline solution for FACS sorting. Alternatively, cell pellets can be resuspended in PBS(-). (OPTIONAL: Cell viability and purity can be checked by flow cytometry using viability dye.)
9) In a 1.5-ml Eppendorf tube prepare the desired volume of lysis buffer by adding the reagents as in section 2.5.2. and then place it on ice. (CRITICAL: For each experiment, the lysis buffer should be made fresh.)
10) Prepare 384-well thin-walled PCR plates for sorting by adding 2 µL of lysis/FACS sort buffer to each well, except NTC-control well(s). (CRITICAL: Maintain the lysis buffer added plates at 4 ℃ using chill blocks)
11) Prepare the FACS instrument with 100/130-micron nozzle size for daily FACS setup, testing, and droplet delay optimization, plate targeting. (CRITICAL: Failure to optimize the droplet breakoff may sort satellite droplet instead of the droplet of interest by placing a charge on the satellite. Follow the FACS manufacturer’s instructions for the droplet stream optimization for timing delay.)
12) Using the FACSDiva software, prepare the gating strategy for doublet discrimination gating to prevent the sorting of cell doublets or multiple cell clumps. Load a small amount of sample into the instrument to confirm the set gating and rearrange the gates if needed. Adjust the voltage of the instrument for each channel if needed.
13) Confirm the FACS setting parameters for single-cell sorting by targeting the plate using 10-µm yellow fluorescent polystyrene microspheres or similar fluorescent beads and observing under a fluorescent microscope for accurate targeting. (CRITICAL: Achieving an accuracy of a minimum 95 % single microsphere sorting is recommended. To obtain this we suggest practice sorts for single microsphere before actual sort day.)
14) Proceed with FACS sorting for a single cell. The overall event rate for 100-micron nozzle is kept at 1000–2,000 events per second on the FACS instrument (minimum 1200 events for 130-micron nozzle setup). Sort 1 cell in each except the Control wells (CRITICAL/OPTIONAL: Final confirmation of single-cell sorting can be performed by sorting a single cell in a slide and observing under microscope.)? TROUBLESHOOTING
15) Seal the sorted plates with MicroAmp Thermo-Seal lid, and immediately proceed with lysis and reverse transcription. PAUSE POINT: Otherwise, immediately freeze the plate on dry ice for storage at −80 °C.
3.4. cDNA synthesis by Smart-seq2 • TIMING: 1 day all steps
We performed cDNA synthesis by using modified Smart-seq2 protocol, previously published by our team. Cell lysis, cDNA synthesis and Nextera XT library preparation can be performed using any of the currently available methods for single cells 39–41. All liquid dispensing steps are performed using the BioCel-1200 system incorporated with Bravo and BioRapTR fluidics systems. For dispensing of master mixes by BioCel we used Half-reaction volumes that have been shown below.
3.4.1. Single cell lysis • TIMING: ~15 min
16) Perform cell lysis on each single cell well by adding lysis mix containing 25 µM Oligo dT (0.25 µL) and 25 mM dNTP mix (0.25 µL) to each reaction mix (see section 2.5.3). To the control wells, add 10 pg/µL UHR (1 µL) to UHR-Control well; nuclease-free water (2 µL) to each NTC-control well; nothing to ERCC spike-in control wells. (CRITICAL: If FACS sorted plates are taken out of -80 ℃ storage, thaw the plates on ice in chill blocks.)
17) Denature by incubating at 72 ℃ for 3 min and immediately putting the plate on ice.
18) Centrifuge at 700 x g for 10 sec at RT to spin down the samples to the bottom of the well. Immediately put the plate back on ice. At this step, the oligo-dT primer is hybridized to the poly(A) tail of mRNA strands.
3.4.2. Reverse transcriptase (RT) reaction • TIMING: 3 hrs
19) Add reagents for RT-reaction as on section 2.5.4 to each well containing 2.50 µL of lysed cell soup (STEP 18) by adding 3 µL of RT-master mix.
20) Perform first-strand cDNA synthesis of RT-reaction in Thermocycler by following reaction cycle:
42 ℃ for 90 min : RT and template-switching
10 cycles of
50 ℃ for 2 min : RNA-secondary structure unfolding
42 ℃ for 2 min : Completion of RT and template-switching
70 ℃ for 15 min : Final heat inactivation of enzyme
4 ℃ hold : Temperature for safe storage.
3.4.3. PCR-preamplification • TIMING: 3 hrs
21) Prepare PCR-preamplification master mix for cDNA synthesis by addition of ISPCR primer as in section 2.5.5.
22) Run PCR-preamplification reaction in a thermocycler by using the following reaction cycle:
98 ℃ for 3 min : Denaturation
21 cycles of
98 ℃ for 20 min : Denaturation
67 ℃ for 15 sec : Annealing
72 ℃ for 6 min : Extension
72 ℃ for 5 min : Final extension
4 ℃ for Infinite : HOLD temperature
PAUSE POINT: PCR run plate can be stored at -20 ℃ for short term or at -80 ℃ for long term storage
3.4.4. PCR Purification of cDNA synthesis product • TIMING: ~45 min
23) Using BRAVO protocol, perform the purification of the cDNA synthesis product by adding AMPure XP beads (1:1 ratio) to the RT-reaction mix from above. Incubate the mix for 5 min at RT and then place it on a magnetic rack for 2 min. Carefully remove the supernatant by pipetting and wash the beads twice with 80 % ethanol (molecular biology grade) for 30 sec. Dry the beads on a magnetic rack for 10 sec. Finally, elute the biotinylated-cDNA with 12.5 µL of Low Tris-EDTA (TE) buffer (10 mM Tris + 0.1 mM EDTA) by incubating for 10 min at RT followed by 2 min on a magnetic rack. (CRITICAL: Low TE buffer and AMPure XP beads should be at RT.)
24) Collect purified cDNA by pulling the supernatant onto a newly labeled thin-wall PCR plate. PAUSE POINT: Seal the plate and store it at -80 ℃. Otherwise, proceed with the QC step for analysis of cDNA quality as in the next step.
3.5. Quality control analysis of purified cDNA • TIMING: 1-4 hrs
(CRITICAL: Quality of purified cDNA library can be analyzed by four methods: Agilent high-sensitivity DNA chips, PicoGreen dsDNA assay, qRT-PCR for expression of housekeeping gene, and qRT-PCR for expression of cell-type specific gene.) Agilent high-sensitivity DNA chips are used for a few randomly picked cDNA samples and analyzed on the 2100-Bioanalyzer system to check the fragment size of DNA. PicoGreen dsDNA analysis is performed on the whole plate to accurately quantify the cDNA concentration in each well. Finally, TaqMan assay is performed in qRT-PCR to check the expression of housekeeping genes such as β-actin (ACTB) to make sure that each well has an eukaryotic cell. (OPTIONAL: TaqMan assay for a known cell-type specific marker gene(s) can be used to confirm the target cell sorted into each well. We didn’t use any neutrophil specific marker as we performed unbiased sorting.)
3.5.1. QC1: Quality check of cDNA library by Agilent high-sensitivity DNA kit • TIMING: 1 hr
25) cDNA library size distribution and quality are checked by Agilent high-sensitivity DNA chips on 2100-Bioanalyzer system for randomly picked cDNA samples from the plate by following the manufacturer's protocol for Agilent high-sensitivity DNA kit.
26) Undiluted cDNA sample (1.0 µL) is loaded on each of the Agilent high-sensitivity DNA chip and then run on the 2100-Bioanalyzer system to obtain the raw data.
27) The sample run data of each chip was analyzed by 2100 Expert software to obtain the electropherogram. Sample free of < 500 bp fragments and showing a peak at ~1.5-2 kb is considered a good library.? TROUBLESHOOTING
3.5.2. QC2: Picogreen dsDNA quantitation assay • TIMING: 2 hrs
28) Quant-iT PicoGreen dsDNA assay kit (Invitrogen, cat. no. P11496) is used for quantification of dsDNA following the manufacturer’s protocol. Working concentration of PicoGreen solution from stock concentration is prepared by 200-fold dilution in TE-buffer (1X) as in section 2.5.6 and 24.50 µL is dispensed in each well of a 384 Black flat bottom plate using BioRapTR.
29) On the other hand, prepare Lambda (λ) gDNA standard of 10 ng/µL working concentration from the provided stock solution in TE-buffer (1X). Prepare λ-gDNA of varying concentrations by serial dilution (10.00 ng/µL, 5.00 ng/µL, 2.50 ng/µL, 1.25 ng/µL, 0.625 ng/µL, 0.3125 ng/µL, 0.15625 ng/µL, and 0.00 ng/µL) to obtain the standard curve, which is used to determine the cDNA concentration of each sample well by plotting the RFU (Relative Fluorescence Unit) value of each sample in the standard curve.
30) To each of the reaction well transfer 0.50 µL of purified cDNA samples using Bravo to obtain a final concentration of 1:50 dilution. To the standard wells, by using a pipette manually load 0.50 µL of Lambda (λ) gDNA standard of varying concentration prepared by serial dilution (see section 2.5.7).
31) Seal the plate, mix the reaction components and centrifuge briefly to bring the reaction mix to the bottom.
32) Incubate for 2-5 min at RT by protecting from light.
33) Read plates in FlexStation 3 or any available Fluorescent microplate reader using standard Fluorescein wavelengths of Ex/Em of 480/520 nm
34) The RFU-values obtained from each sample well is plotted against the standard curve generated from the lambda-DNA standards (obtained by serial dilutions) to obtain the cDNA concentration of each single-cell well and the control wells (i.e. NTC, ERCC and UHR controls) by using the SoftMax Pro Software for FlexStation 3 (Molecular Devices).
35) The SoftMax Pro software generated file is saved in .txt format. The cDNA concentration for each sample well is then incorporated in the final project template file in .xlsx format. Samples with DNA concentration > 0.30 ng/µL are considered good suitable for downstream processes.? TROUBLESHOOTING
3.5.3. QC3: qRT-PCR for housekeeping gene expression • TIMING: 4 hrs
36) For qRT-PCR TaqMan assay, first dilute the cDNA sample to 1:10 in a new 384 well FrameStar plate by adding 9 µL Low TE buffer or nuclease free water to 1 µL of cDNA using Bravo.
37) Prepare required volume of qRT-PCR master mix as in section 2.5.8 to be dispensed to each well of the PCR-plate by BioRapTR. (CRITICAL: Consider the “dead volume” of BioRapTR while calculating the total required volume of qRT-PCR Master mix.)
38) Dispense 7.50 µL RT-Master mix with BioRPTR using designated Reservoir and Tip to each well.
39) Using BRAVO, add 2.5 µL of diluted cDNA template of each sample for a total reaction volume of 10 µL/well. For NTC-control wells, added 2.5 µL of nuclease-free water.
40) Load the reaction plate on qRT-PCR machine (QuantStudio 6 Flex Real-Time PCR system) and run the following reaction cycle:
95 ℃ for 2 min : Denaturation
50 cycles of
95 ℃ for 10 sec : Denaturation
60 ℃ for 30 sec : Annealing
4 ℃ for Infinite : HOLD temperature
41) On completion of the qRT-PCR reaction cycles, analysis of the generated raw data is done by QuantStudio Real Time PCR Software V1.3. The "CT Settings” under the "Analysis” is changed from its “Default Settings” by changing the ‘Threshold' to 0.01 and 'Baseline Start and End' to 2 and 10 respectively. Then “Analysis Settings" is applied to obtain the final Ct-values.
42) The Ct-values are then exported in the .XLS format to be incorporated in the final project template file. Samples with Ct-values < 35 is considered good quality and suitable for downstream processes.
3.5.4. QC4: qRT-PCR for target cell specific gene expression • TIMING: 4 hrs
(OPTIONAL: Expression of genes known to be expressed in the target cell sorted can be checked by qRT-PCR to confirm the single cell sorted in each well.)
43) Taqman assay or preferred qRT-PCR method targeting the marker gene specific to the target cell sorted for the study can be performed to verify the single cell sorted in each well
3.5.5. cDNA-library plate preparing by HitPicking of QC-Pass wells • TIMING: 2 hrs
44) The cDNA concentration obtained from PicoGreen assay (From STEP 35) along with the Ct-values for ACTB expression obtained from qRT-PCR Taqman Assay (From STEP 42) for each sample well in sample plate is pasted on the project template file. Project template file is an excel file prepared to keep track of each sample well of each sort plate.
45) The samples having cDNA concentration > 0.3 ng/µL and ACTB Ct-values < 35 are considered double QC-Pass and selected for Hamilton transfer to a new cDNA-library plate.
46) For Hamilton transfer, a “Hamilton input file” in .CSV format is generated for each library plate of each donor (viz. H-SN1_Lib#1 etc.). This file is loaded in the “VENUS” software and Hamilton transfer protocol is run after placing the desired tips and plates at their designated location set on the protocol.
47) On completion of the Hamilton transfer to combine HitPicked cDNA-library plate from two/three single-cell cDNA plates, the plates are sealed, barcoded and stored at -80 ℃ for downstream processes.
3.6. Illumina Nextera XT Library preparation of HitPicked cDNA-library plates:
Illumina Nextera XT library is prepared for the HitPicked cDNA library plate by using ‘Nextera XT DNA library preparation kit’ and each sample is barcoded by using ‘Nextera XT index kit Set A and Set D’ following the manufacturers protocol. We used 1/8th reaction protocol for automated/robotic dispensing system, where 1/8th the volume of each reagent is used as mentioned in manufacturers protocol for 96-well reaction plate. The required target DNA quality for Nextera Library preparation is 1 ng of input DNA with 260/280 ratio of 2.0 - 2.2
3.6.1. Normalization of cDNA library plate • TIMING: ~1 hr
48) Before starting the Nextera XT 1/8th reaction protocol, ‘Normalized cDNA Library’ plate is prepared to obtain 0.2 ng/µL cDNA concentration in all wells.
49) cDNA library plate stored at -80 ℃ is taken out and thawed on a chill block in ice.
50) From each sample well 1 µL of cDNA is transferred to a new Framestar 384-plate by BRAVO and the desired volume of low TE-buffer is dispensed by BioRapTR to obtain 0.2 ng/µL cDNA concentration in each well. Care is taken so that total volume per well should not exceed 100 µL. If the calculated volume for any well exceeds 100 µL, the final dispensing volume is calculated for 100 µL.
3.6.2. Nextera XT Tagmentation reaction • TIMING: 10 min
51) 0.625 µL of diluted cDNA (0.2 ng/µL) from cDNA normalization plate is added to 1.250 µL of Tagment DNA Buffer (TD, 2X) and 0.625 µL of Amplification Tagment Mix (ATM) in a Framestar 384-well microplate to obtain 2.5 µL total tagmentation reaction mix volume.
52) The plate is sealed, mixed by brief centrifuge and loaded on the thermocycler to run the tagmentation reaction by incubation at 55 °C for 10 min.
53) On completion of the reaction, immediately add 0.625 µL of NT buffer to neutralize the Tagmentation reaction to obtain 3.125 µL Total Neutralized Tagmentation volume/well.
3.6.3. Nextera XT PCR reaction with Set A and Set D barcoding kits • TIMING: 1 hr
54) To the 3.125 µL of Tagmentation volume, 1.875 µL NPM PCR master mix and 1.250 µL of Index Primer mix (0.625 µL Index Primer i5 + 0.625 µL Index Primer i7) is added to obtain total volume of 6.25 µL Nextera PCR reaction/well.
55) Seal and centrifuge FrameStar Plate at 4 °C, 500 x g (2,000 RPM) for 30 sec to mix, keep on ice till running the thermocycler reaction.
72 °C for 3 min : Extension
95 °C for 30 sec : Denaturation
16 cycles of
95 °C for 10 sec : Denaturation
55 °C for 30 sec : Annealing
72 °C for 60 sec : Extension
72 °C for 5 min : Final extension
4 °C for Infinite : HOLD
PAUSE POINT: Seal the plate and store at -80 °C until ready for library purification and cleanup.
3.6.4. Nextera XT Library purification and cleanup • TIMING: ~45 min
56) Purify each sample individually as is Step 23-24 but use 0.9:1 ratio of AMPure XP beads to Nextera library (i.e. 5.625 µL beads + 6.25 µL of Nextera Library). Elute with 6.25 µL of Low TE buffer into a new FrameStar plate "Purified Nextera XT".
PAUSE POINT: Seal the plate and store at -80 °C until ready for PicoGreen QC or normalization of the purified library.
3.6.5. QC5: Nextera XT Library QC by PicoGreen assay • TIMING: 2 hrs
57) Use 1 µL of the purified Nextera XT reactions for Picogreen dsDNA assay as in Step 28-35.
3.6.6. Normalization of Nextera XT Library plate • TIMING: ~1 hr
58) Prepare 1.0 ng/µL normalization plate of purified Nextera library based on the PicoGreen quantification above.
59) Using BioRaPTR dispense the desired amount of Low-TE buffer to a new Framestar plate and then using BRAVO to add 1 µL of purified Nextera XT library sample to obtain 1.0 ng/µL purified Nextera XT sample in each well.
3.6.7. 16 sample pooling of NexteraXT samples for MiSeq run • TIMING: 30 min
(OPTIONAL: As the sequencing of NovaSeq run for 384-plex pool library is expensive, we sequenced randomly picked 16-plex pool library from each NexteraXT library plate in MiSeq-Nano to confirm the sequence quality, determine the coverage needed and required depth of the transcriptome. We used Illumina MiSeq Reagent kit v2 (300 cycle) for the MiSeq-Nano low output run.)
60) Pool 16 samples (from Step- 59) by pipetting 3 µL of all normalized NexteraXT samples (1.0 ng/µL) for a 3 ng pool into a 1.5 mL Eppendorf LoBind tube.
61) Reverse pipette to determine the total volume and then add 90 % of that volume AMPure beads to purify the MiSeq-pooled library as in Nextera XT library cleanup on Step 56.
62) Elute with Low-TE buffer using 10-fold lower volume than the original volume of pooled library determined by reverse pipetting.
63) For QC check on Agilent high-sensitivity DNA chips, prepare 1:1 dilution (take out 1 µL), 1:10 dilution (1 µL library + 9 µL Low TE), and 1:20 dilution (2 µL of 1:10 dilution + 2 µL of Low-TE)
64) Run each of the 3 samples in triplicate on Agilent high-sensitivity DNA chip. Calculate the average fragment size and the average pool concentration in pmol/L (pM) and nM of all replicates from DNA chip run report.
PAUSE POINT: Store the MiSeq-pooled library at -20 °C until ready for sequencing run.
3.6.8. 384 sample pooling of NexteraXT samples for NovaSeq run • TIMING: 30 min
65) For each 384-well library plate, combine all 384 normalized Nextera XT libraries from each well to a single well by using BRAVO. Pipette the 384-plex pool to an Eppendorf tube and label the tube with sample and library name (e.g. H-SN1_Lib#1, H-SN2_Lib#1, and H-SN3_Lib#1 in this study).
3.6.9. Pooled Nextera XT Library Cleanup • TIMING: ~45 min
66) Clean the 384-plex pool Nextera XT library by AMPure XP bead purification as in Step 23-24 using manual protocol for fewer pooled library sample tubes. BRAVO protocol for AMPure XP bead purification can be used for more samples.
3.6.10. QC6: QC check of 384-plex Nextera XT Library (Agilent DNA Chip) • TIMING: 1 hr
67) Use 1 µL of the 384-plex pool NexteraXT Library samples to check the quality on 2100-Bioanalyzer using Agilent high-sensitivity DNA chips. The DNA concentration of the sample is assumed to be within the range recommended by Agilent for the high-sensitivity DNA chips.
68) The Bioanalyzer report for each 384-plex pool library is saved and needed to submit samples to the sequencing core.
PAUSE POINT: The Pooled library can be stored at -20 °C for short term and at -80 °C for long term till ready for sequencing run.
3.6.11. QC7: KAPA Library Quantification Kits - Illumina • TIMING: 2-3 hrs
69) Calculate and prepare volumes enough for 3 replicates of NTC, each sample dilutions, and each standard.
70) Six pre-diluted DNA Standards of concentration 20, 2, 0.2, 0.02, 0.002, 0.0002 pM respectively are provided in the kit.
71) Before starting, prepare four different dilution sets of the pooled NexteraXT library (from Step 66) by adding required volume of dilution solution (10 mM Tris-HCL + 0.05 % Tween 20) as in section 2.5.9.
72) Prepare qPCR master mix as in section 2.5.10 by combining the 1 mL of Illumina Primer Premix (10X) and the 5 mL bottle of KAPA SYBR Fast qPCR master mix (2X) provided in the kit after thawing properly. Vortex briefly to mix well and store at -20 °C till ready to use.
73) For half reaction volume, add 6 µL of qPCR master mix and 4 µL of sample or standards to appropriate well. Add 4 µL nuclease free water to NTC wells
74) Seal plate, gently vortex to mix and spin down sample to bottom of the well. Protect plate from light until ready to run.
75) Run the sample plate on QuantStudio 6 Flex or any other qPCR machine by selecting the “Standard Curve” experimental method and “SYBR Green” detector. Run the instrument in “Fast” mode using the following thermocycler protocol:
95 °C for 5 min : Denaturation
35 cycles of
95 °C for 20 sec : Denaturation
60°C for 45 sec : Annealing
4 °C for Infinite : HOLD
76) On completion of qPCR run, analyze the data and evaluate the Slope and R2. If the auto set Ct-threshold if the acceptable range of slope (-3.58 to -3.10) and/or R2 (~0.99) is not obtained, manually adjust Ct by setting threshold of 0.2 and set start cycle to 2 and end cycle to 3.
77) The average Ct value of each DNA Standard is plotted against its known concentration (pM) to generate a standard curve which is used to determine the concentration (pM) of diluted libraries. Finally, the working concentration of each library is calculated from the concentration of diluted libraries.
78) To further check the fragment size, the qPCR amplified product from three replicate wells were combined and ran on 1.2 % E-Gel (Invitrogen, cat. no. A03076) for 30 min using 1 kb plus ladder (Invitrogen, cat. no. 10787018) and 6X loading dye (Promega, cat. no. G190A).
79) On verification of the quality and fragment size of the pooled NexteraXT library samples, proceed to the cDNA sequencing step.
3.7. cDNA Sequencing
3.7.1 cDNA Sequencing: kit selection, run parameters, and yield • TIMING: ~24 hrs
80) The purified pooled-NexteraXT library is subjected to paired-end sequencing on a suitable Illumina NGS platform (MiSeq, HiSeq 2500, NextSeq 500, and NovaSeq 6000) with the aim to generate 1-2 million reads per sample having a read length of 100-150 bases. The sequencing data generated by the HGS platform is in fastq format.
The following sections have been briefly explained in this paper. For details on “RNA-seq analysis” of the fastq data files, please refer to Step 26 of previous publication from our group 24.
3.7.2. RNA-seq analysis: sequence quality assessment and preprocessing • TIMING: Variable
81) Sequence quality assessment: Sequence quality is assessed by evaluating the fastq sequence files (from Step 80) from each cell (i.e. single cell saliva neutrophil) using the fastQC tool for sequence yield, base quality, GC profile, k-mer distribution, contamination and other desired parameters.
82) Sequence duplication: Sequence duplication is determined in the input data. Tools such as fastx_collapser are used to calculate the absolute number of identical reads (i.e. duplicates) in the input sample fastq sequences (from Step 80). Use correct base quality score offset (-Q). Process multiple files by repeating each sequence file at a time, as the program accepts only one sequence file as input.
83) Sequence trimming: Sequence trimming of input paired-end fastq reads (from Step 80) is performed by Trimmomatic program to remove adapter/primer sequences and low-quality end bases.
3.7.3. RNA-seq analysis: sequence mapping and gene expression analysis • TIMING: Variable
84) Prepare the reference genome: Prepare the reference genome index for alignment using the build function in HISAT2 program and the reference genome fasta file. Here we used GRCh38 downloaded from Ensembl /Gencode.
85) Calculating expression values: Calculate the gene expression values (transcripts per million or TPM) by mapping the paired-end reads that passed trimming (from Step 83) to the reference index using HISAT2 and then evaluating the alignments using StringTie to estimate levels of expression per gene models in the annotation file. Here we used gencode.v25.annotation.gtf.
86) Calculate and plot overall mapping statistics: Calculate the number of reads that are mapped to the genome, to the ERCC spike-in transcripts as well as that remained unmapped using SAM tools.