Extended Data Fig. 11: FACS-assisted bulk RNA-seq of cortical PV interneurons.
From: Calcium-permeable AMPA receptors govern PV neuron feature selectivity
a, Overview of the workflow isolating and analyzing mouse cortex PV interneuron mRNA expression. Fixation-capture single cell RNA recovery-seq (FICSR-seq) was used to recover PV interneurons without substantial loss of PV cells during dissociation. After brain slicing, enzymatic dissociation was followed with fixation in 4% PFA and mechanical dissociation. GFP+/DRAQ5+ cells were isolated using FACS and were treated with proteinase K before RNA extraction which removes RNA-binding proteins and increases the yield of intact RNA. The resulting mRNA was sequenced with paired-end Illumina sequencing and analyzed for differential gene expression. b, Representative gating diagrams and FACS flowchart. DRAQ-5 was used to sort nuclei-containing cells from debris, and singlets are further sorted into GFP+ and GFP- cells. c, d, Bulk RNA-seq reveals ~100-fold enrichment of Pvalb mRNA in GFP+ vs GFP- cell samples (from n = 16/2 mice, mean ± SEM), validating FACS-based isolation of PV interneuron population. TPM stands for transcripts per million. e, Differential gene expression in PV-Cre;lsl-eGFP-GluA2 mice vs. PV-Cre;lsl-eGFP mice (n = 7/9 mice). Gria2 transgenic overexpression is observed (Padj = 4.63×10−9, Benjamini-Hochberg correction), together with other regulated genes (red).
