Mouse cortex PV interneuron transcriptome changes assessed with FACS-assisted bulk RNA-seq

To investigate the novel link between CP-AMPARs and excitability, we assessed global PV interneuron transcriptome changes with FICSR-seq (Fixation-Capture Single Cell RNA Recovery-seq) on forebrain PV interneurons. FACS-assisted PV interneuron bulk RNA-seq of PV-Cre;lsl-eGFP-GluA2 mice and PV-Cre;lsl-eGFP controls showed no expression changes in 278 out of 279 genes comprising the major classes of ion channels and excitatory/inhibitory synapse proteins. This lack of expression changes suggests a post-transcriptional regulation of intrinsic and extrinsic (synaptic) excitability. The exception was GluA2 mRNA, which was expressed ~2 fold compared to control PV interneurons, in agreement with protein measurements. GluA1, although downregulated at protein level, was unchanged at the mRNA level. These transcriptomic results suggest that the substantial changes in PV interneuron excitability after CP-AMPAR removal are not supported by changes in gene expression but likely reflect post-transcriptional regulation. We developed a technique to preserve neurons after dissociation, fixation-capture single-cell RNA recovery-seq (FICSR-seq), which recovers PV interneurons vulnerable to dissociation at native cell frequencies. We cut brain slices from adult PV-Cre;lsl-eGFP and PV-Cre;lsl-eGFP-GluA2 mice (113.1 ± 11.6 days old) in NMDG cutting solution + Trehalose and diced them into small pieces <1 mm3. Extracellular proteins were digested with pronase (2 mg/ml; 8 U/µl) at 34-37°C, after which the slice pieces were fixed in 4% PFA in PBS (with 0.1 U/ml RNase inhibitor, Promega) for 15 mins and dissociated into single cells through careful trituration. We filtered the single cells through a 40 μm filter, labeled them with the cell-permeable nuclear dye DRAQ5 (1:1,000 dilution) to identify nuclei-containing cells, and then subjected them to FACS. DRAQ5+/GFP+ or DRAQ5+/GFP-GluA2+ cells were sorted, and over 20K cells were collected per mouse cortex to provide extensive coverage of low-expressing PV interneuron transcripts. We treated the fixed cells with Proteinase K before RNA extraction (RecoverAll Total Nucleic Acid Isolation Kit for FFPE, Thermo Fisher Scientific) to liberate RNA from protein-protein and protein-nucleic acid crosslinks generated by fixation. We prepared cDNA libraries from PV-Cre;lsl-eGFP and PV-Cre;lsl-eGFP-GluA2 mouse brains (NEBNext Ultra RNA Library Prep Kit for Illumina, NEB) from RNA enriched with mRNA through bead-based polyA selection. cDNA libraries were barcoded and sequenced together on an Illumina Hiseq 2500 sequencer, generating 150-bp paired-end reads. We processed RNA-seq reads with bcbio-nextgen v1.2.3, aligning to GRCm38 with the STAR aligner and quantifying counts per gene with Sailfish using the Ensembl annotation. We used DESeq2 to analyze differential expression.