SRA

Primary afferent pathophysiology is thought to be a key driver in painful conditions, with dorsal root ganglia (DRG) neurons being well described for their role in driving both acute and chronic pain. These neurons encompass a diverse collection of subtypes that are grouped by various factors, each intrinsically related. These include size, myelination, conduction velocity, projection patterns, end organs, and general function. More recently, these subpopulations have been well documented through single cell and single nuclear RNA-seq in mice and human. This diversity is lost during bulk RNA-seq, due to the consolidation of all subtypes together. In single cell datasets, pseudo-bulk samples can be generated for each cluster, but this relies on a well-defined clustering that can be lost after nerve injury. As such, changes at a subtype level remain unclear in painful states. Here, we address this gap through the deep transcriptional profiles of multiple murine DRG populations in acute and chronic pain states while considering sex differences. We have studied the molecular changes in five populations: Scn10a-expressing DRG, peptidergic and non-peptidergic nociceptors, as well as C-LTMRs and Ntrk2-expressing A-LTMRs. In a naive state, we find subtype specific sexual dimorphism in a small number of genes. This does not translate to a strong interaction of sex and injury, as the injury response seems to be consistent across sexes at the neuronal transcript level. We also see both stereotypes and unique subpopulation signatures in injured states after nerve injury at both an acute (3 day) and chronic (4 week) timepoint, with notable changes in C-LTMR and NP populations, as well as a distinct transcriptional program in AB-RA + Ad-LTMRs by 4 weeks compared to the other subtypes in the study. This data has been deposited in a searchable database to increase accessibility at https://livedataoxford.shinyapps.io/drg-directory/. Overall design: Here, we perform comparative gene expression profiling analysis of bulk RNA-seq of murine sensory neuron subtypes at two timepoints after nerve injury (SNI), with sex differences. Sensory neurons were labelled through previously available transgenic lines crossed to fluorescent reporters for FACS, allowing for bulk purified (via FACS) subtype-specific samples. Once all samples were collected, samples were thawed on ice, vortexed, and randomized into a 384-well plate. Non-directional libraries were prepared together using NEB Ultra low/Smarter library prep, as per manufacture instructions by the Oxford Genomics Centre at the Wellcome Trust Centre for Human Genetics. Libraries were amplified (21 cycles) on a Tetrad (Bio-Rad) using in-house unique dual indexing primers (based on Lamble et al. 2013). Individual libraries were normalized using Qubit, and the size profile was analysed on the 2200 or 4200 TapeStation before pooling together accordingly. The pooled library was diluted to ~10 nM for storage. The 10 nM library was denatured and further diluted prior to loading on the sequencer. Sequencing was performed over three independent runs, and merged after quality control. Paired end sequencing was performed using a NovaSeq6000 platform using the S2/S4 reagent kit v1.5. Samples were sequenced with a 150~bp read length, at a depth of 30 million reads per sample.