TITLE: Protocol for RNAseq analysis of identified gastric vagal afferent neurons
AUTHORS: Martin Stebbing, Juan C Molero

[DESCRIPTION]
This protocol describes methods to dissociate nodose ganglion neurons from rats, identify and specifically collect neurons previously labeled using retrograde tracing, and then perform short read RNAseq analysis on pooled samples of small numbers of neurons or single neuronal cells. The parameters of the analysis are designed to allow deep sequencing (pooled cell samples, high numbers of reads per sample) to enable robust identification of genes expressed at low copy numbers that are important for neuronal function, such as ion channels and G-protein coupled receptors. It also includes a basic description of the sorts of differential expression analysis that can be performed on sequencing/transcriptomic data of this type.

[BEFORE_START]
Before conducting any experiments using animals, ethics approval should be sought and obtained in accordance with local legal requirements and ethical standards as outlined in national and international guidelines for the use of animals in research.

Be aware that this protocol includes several critical steps that require some skill, practice, and care, including difficult dissections that must be done quickly, trituration steps and cell sampling that require skill, practice, and optimization under specific local conditions, as well as steps that require an RNAse free and uncontaminated workspace.

Dissociation of neuronal cells is a difficult process that requires skill. If not optimized, cell yield will be low, and cell viability will be compromised. Poor sample quality will result in wastage of expensive reagents and poor outcomes from expensive sequencing runs. Care should be taken to optimize conditions and practice skills before experimental samples are collected and processed for sequencing.

[GUIDELINES]
All animal procedures used in these protocols were approved by the Animal Experimentation Ethics Committee of the Florey Institute of Neuroscience and Mental Health and designed and performed in accordance with guidelines of the National Health and Medical Research Council of Australia.

[STEPS]
SECTION: Tissue harvesting
1. Vagal neurons innervating either the muscle layers or the mucosa in various stomach regions in Sprague Dawley rats (RRID: RGD_10395233), are identified using injections of fluorescent beads as described in a separate protocol (see below)

 

SECTION: Tissue harvesting
2. Rats that have received tracer injections into the stomach 7-14 days previously are euthanized by overdose with Pentobarbital (100 mg/kg i.p.)  

SECTION: Cell Collection
14. Neurons are viewed on an inverted fluorescence microscope with red and green filters and labeled neurons identified by the characteristic red fluorescent cytoplasmic puncta within their cytoplasm. These puncta are extremely bright and extremely small, so that focusing up and down causes a characteristic ‘sparkling effect’ as the beads come in and out of focus.
SECTION: Nodose Ganglion dissociation
5. Left (LNG) and/or right (RNG) nodose ganglia are digested separately with 2 mg/mL collagenase type I (Worthington, Cat no LS004196/7) in L-15 medium at 37oC with orbital shaking (120 rpm) for 45 minutes.  

SECTION: Nodose Ganglion dissociation
7. Samples are further digested with 2 mg/mL collagenase type I and 0.075% Trypsin-EDTA (Sigma, Cat noT4049) in L-15 medium at 37oC with orbital shaking (120 rpm) for 45 minutes.  
  

SECTION: mRNA isolation, amplification cDNA purification and quality analysis
20. mRNA amplification and cDNA synthesis of sampled dissociated neurons is performed using SMART-Seq HT kits (Takara Bio USA, Inc., Cat no634456) according to the manufacturer instructions.   

SECTION: mRNA isolation, amplification cDNA purification and quality analysis
21. Briefly, lysis buffer containing RNAase inhibitors is added while cells are still frozen. One step PCR reactions containing both first strand synthesis SMARTscribe reverse transcriptase and SeqAmp DNA polymerase are set up with the following protocol:

1) 42oC for 90 min; 
2) 95oC for 1 min;
3) 98oC for 10 sec;
4) 65oC for 30 sec;
5) 68oC for 3 min;

(Steps 3-5 for 14 cycles [pooled cell samples] or 16 cycles [single cell samples] depending on the sample type);

6) 72oC for 10 min.
SECTION: cDNA fragment library generation and sequencing
23. These steps are performed by a service provider, namely the Monash Medical Genomics facility according to our requested protocol. Samples are transported on dry ice directly to the facility. 
SECTION: RNAseq analysis
30. Differential Expression Gene (DEG) analysis is performed using the open source, web-based Galaxy platform.
SECTION: Tissue harvesting
4. The stomach is also dissected out, opened flat, and fixed in 4% PFA in PBS overnight at 4oC for later examination of injection sites (see protocol quoted in step 1)
SECTION: Tissue harvesting
3. Nodose ganglia are immediately dissected out and placed in ice-cold Leibovitz’s L-15 medium (Gibco/Thermo Fisher, Cat no21083-027). Connective tissue around the ganglia is removed, and a longitudinal slit is made in the capsule with a scalpel blade.  

SECTION: Nodose Ganglion dissociation
6. The resulting samples are spun down at 400 x g for 5 min at room temperature.
SECTION: Nodose Ganglion dissociation
8. Samples are next spun down at 1000 x g for 5 min at 4oC and washed in L-15 medium containing 100 μunits/mL DNAase type I (Sigma, Cat no D-4527).  

SECTION: Nodose Ganglion dissociation
9. Samples are again spun down at 1000 x g for 5 min at 4oC. 
Ganglia are washed for the second time in L-15 medium containing 100 μunits/mL DNAase type I  

SECTION: Nodose Ganglion dissociation
10. Nodose ganglion samples are spun down at 1000 x g for 5 min at 4oC. 
SECTION: Nodose Ganglion dissociation
11. Nodose ganglion samples are resuspended in L-15 medium containing 100 μunits/mL DNAase type I and triturated with flame-polished glass Pasteur’s pipettes coated with 1% bovine serum albumin. Ganglia are passed up and down the stem of pipettes with decreasing inner diameter until achieving a homogeneous cell suspension.
SECTION: Nodose Ganglion dissociation
12. Nodose ganglion cell suspensions are spun down at 1000 x g for 5 min at 4oC.
SECTION: Nodose Ganglion dissociation
13. Dissociated nodose ganglia cells are resuspended in L-15 medium without enzymes and placed on coverslip bottom 35 mm culture dishes (Fluorodish, WPI). Cells are left to settle down for 20 minutes before manual cell picking.  

SECTION: Cell Collection
15. Only clearly isolated single cells with a defined plasma membrane are chosen for sampling. The area around the cell is first cleared of debris and other cells, using a small diameter ‘sweeping’ pipette attached to a micromanipulator, and the position of each cell to be sampled is recorded using the vernier scale on the microscope stage.
SECTION: Cell Collection
16. After applying positive pressure to it via an attached tube and valve, a sampling pipette beveled at 45 degrees is introduced into the bath with the bevel parallel to the surface of the dish using a micromanipulator. The pipette aperture is placed over the cell to be sampled before a very slight positive pressure is applied to pull the cell and only the cell into the pipette.
SECTION: Cell Collection
17. When pooled cell samples are being collected, up to 12 labeled cells are collected into the same pipette in a similar fashion. With any indication that debris or unlabeled cells have been inadvertently sampled, the sample should be discarded.
SECTION: Cell Collection
18. Unlabeled control cells are collected in a similar way, except that clearing of surrounding cells is not performed before sampling.
SECTION: Cell Collection
19. The collected cell(s) is/are expelled from the pipette with slight positive pressure into an RNAse free microfuge tube and the pipette tip is broken within the tube to ensure any adherent cells are also collected.  After a brief spin to aggregate the sample, the volume of solution collected is estimated and the sample is snap frozen on dry ice and kept at -80oC until use. 
SECTION: mRNA isolation, amplification cDNA purification and quality analysis
22. cDNA is purified using a magnetic bead kit (Nucleomag NGS clean-up and Size Select, Macherey-Nagel GmbH & Co., Cat no 744970.5) according to the manufacturer instructions. cDNA quality is assessed by loading purified cDNA into High sensitivity D5000 screen tapes (Agilent Technologies, Cat no 5067-5592) and analyzing them using a 2200 Tapestation system. cDNA concentration is determined using Qubit dsDNA HS Assay kits (Invitrogen, Cat no: Q32851) according to manufacturer instructions.    

SECTION: cDNA fragment library generation and sequencing
24. Briefly, all samples are quantified again at the facility using Qubit, and 600 pg of amplified cDNA is used for library preparation as recommended by the SmartSeq protocol.
SECTION: cDNA fragment library generation and sequencing
25. Apart from this, the samples are processed according to the standard protocol using the Illumina Nextera XT DNA Library Kit (Ref Guide 15031942)   

SECTION: cDNA fragment library generation and sequencing
26. The libraries are next QC’d by Qubit and Bioanalyzer. They should be similar with a mean size of ~360 bp at a concentration of 15 ng/uL. Prior to sequencing libraries are also quantified by qPCR and size adjusted qPCR concentration is used for sequencing.
SECTION: cDNA fragment library generation and sequencing
27. Library pools are prepared so that both control and labeled cell samples are run together with between 12 and 150 barcoded samples combined in each sequencing run. This sequencing design should yield around 30-40 million reads per pooled cell sample or 5-10 million reads per single cell sample (approximately 400-500 million, or 1,100 million total reads depending on the sequencer), so that expression of low copy number genes such as GPCRs can be quantified. 
SECTION: cDNA fragment library generation and sequencing
28. For sequencing, denatured libraries at a loading concentration of 2.0pM (NSQ 550) or non-denatured libraries at a loading concentration of 1000 pM (NSQ 2k) are used and clustering is performed onboard for each high output sequencing run (NextSeq v2.5 75bp runs on an NSQ550 sequencer or v3.0 100 bp runs on an NSQ2k P3 sequencer) together with 1% PhiX control. 
SECTION: cDNA fragment library generation and sequencing
29. Criteria for success include a high number of resulting reads per sample, and >90% of reads with >Q30, PhiX sequencing control detected with error rate <0.5%, Phasing <0.4  and Prephasing <0.2.
SECTION: RNAseq analysis
31. Raw fragment sequencing data (fastq files) is mapped to the latest rat genome assembly (mRatBN7.2, Feb 2021, Sanger Institute) using the alignment tool HISAT2. 
SECTION: RNAseq analysis
32. The number of reads mapped to each gene is determined with the light-weight read counting program Featurecounts. 
SECTION: RNAseq analysis
33. A single count matrix containing all samples is generated and the differential gene expression of nodose neurons projecting to any two different stomach locations is determined by the Edge R method. Counts are normalized by the trimmed mean of M (log2 fold change between the two samples) values. A threshold of 1 count per million reads is used to filter out low count expression genes when using the whole mRatBN7.2 genome assembly. No low count filter is applied when DEG analysis is performed on smaller subsets of genes from mRatBN7.2 (i.e. GPCRs, ion channels, etc…).