High-throughput mapping of single-neuron projection and molecular features by retrograde barcoded labelling [bulk RNA-seq]

Deciphering patterns of connectivity between neurons in the mammalian brain is a critical step toward understanding brain function. Conventional imaging based neuroanatomical tracing methods identify area-to-area or sparse neuron-to-neuron connectivity patterns, but with extremely limited throughput. Recently developed barcode-based connectomics methods can efficiently map large numbers of single-neuron projections, but linking these data to single-cell transcriptomics remains a challenge. Here, we established a retro-AAV barcode-based multiplexed tracing method called MERGE-seq (Multiplexed projection neuRons retroGrade barcodE sequencing), which is capable of simultaneously characterizing the projectome and transcriptome at the single neuron level. We uncovered dedicated and collateral projection patterns of ventromedial prefrontal cortex (vmPFC) neurons to five downstream targets (AI, DMS, BLA, MD and LH). We found that projection-defined vmPFC neurons are molecularly heterogeneous, which are composed of different neuronal subtypes. We further identified transcriptional signatures of various dedicated and bifurcated vmPFC neurons, and verified Pou3f1 as the marker gene of neurons sending collateral axons to DMS and LH. Finally, we fitted our single-neuron connectome/transcriptome data into a machine learning-based model and revealed groups of genes that were predictive of certain projection pattern. In summary, we have developed a new multiplexed technique whose paired connectome and gene expression data can help reveal organizational principles that form neural circuits and process information. Overall design: Male adult C57BL/6 mice (8 weeks of age) were anesthetized intraperitoneally using pentobarbital sodium (10 mg/mL, 120 mg/kg b.w.) and unilaterally injected with rAAV-EGFP-BARCODE virus into five projection targets simultaneously. Barcode 0 sequences representing the AI target is: CTGCACCGACGCATT; barcode 1 sequences representing the DMS target is: GAAGGCACAGACTTT; barcode 2 sequences representing the MD target is: GTTGGCTGCAATCCA; barcode 3 sequences representing the BLA target is: AAGACGCCGTCGCAA; barcode 4 sequences representing the LH target is: TATTCGGAGGACGAC. 3 mice were performed scRNA-seq without FAC-sorting, cell pellets were resuspended and 48,000 cells were loaded into 3 lanes to perform 10X Genomics sequencingperformed scRNA. 3 mice with sorting and pooled together for scRNA-seq. Chromium Single Cell 3' Reagent Kits (v3) were used for transcriptome library preparation (10X Genomics). Libraries were sequenced on an Illumina Novaseq 6000 system. Parallel PCR reactions were performed containing 50 ng of post cDNA amplification reaction cleanup material as a template. P5-Read1 (AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTC) and P7-index-Read2-EGFP (CAAGCAGAAGACGGCATACGAGATAGGATTCGGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTGgCATGGACGAGCTGTACAAG) (200 nM each) were used as primers with the NEBNext Ultra II Q5 Master Mix (NEB, Cat #M0544L). Amplification was performed using the following PCR protocol: (1) 33°C for 1 min, (2) 98° for 10 s, then 65°C for 75 s (20-24 cycles), (3) 75°C for 5 min. Reactions were re-pooled during 1X SPRI selection (Beckman, Cat #B23317), which harvested virus projection barcodes library. 431-437bp (with 120bp adaptors) projectome libraries were sequenced using Illumina HiSeq X Ten.