Cfx96

The hippocampus (both dorsal and ventral) was dissected on dry ice and homogenized. DNA/RNA were extracted (Qiagen Inc., Valencia, Calif., USA) and stored at −80°C following quantification and analysis of nucleic acid quality using spectrophotometry (NanoDrop 2000). Reverse transcription was performed using a cDNA synthesis kit (Qiagen) on RNA, and cDNA was amplified by real-time PCR (Bio-Rad CFX96) with Taqman probes (Applied Biosystems) to target bdnf total mRNA (exon IX) and exon I- and IV-specific transcripts. Tubulin was used as a reference gene based on analysis showing no differences in tubulin expression between neonatal treatment groups in these experiments. All reactions for each gene target and reference were run in triplicate. Product specificity was verified using gel electrophoresis.

cDNA synthesis was performed on RNA purified from sorted GC and soma using SuperScript IV (ThermoFisher). qPCR analysis was performed on a BioRad CFX96 using the TaqMan Gene Expression Assay (Applied Biosystems, ThermoFisher) according to the manufacturer’s protocol. The following TaqMan probes were used: Rplp0 (#4453320; Mm00725448_s1), Rpl18a (#4448892; Mm04205642_gH), Rpsa (#4448892; Mm00726662_s1), Rps24 (#4448892; Mm01623058_s1), Rack1 (Gnb2l1) (#4448892; Mm01291968_g1), Eef1b2 (#4448892; Mm00516995_m1), Eef1g (#4448892; Mm02342826_g1), Cdkn1b (#4453320; Mm00438168_m1), Hspa5 (#4453320; Mm00517691_m1), Ppib (#4453320; Mm00478295_m1), Tubb2b (#4448892; Mm00849948_g1), Actb (#4453320; Mm02619580_g1), Gap43 (#4448892; Mm00500404_m1). Relative abundance in each sample was normalized by the mean expression of all transcripts tested, and enrichment is reported as the mean of GC/soma ratios across 3 biological replicates from independent litters. Sample order was randomized between replicate experiments. Agreement between RNAseq and qPCR enrichment was assayed by calculating R², the square of Pearson’s r, from log2-transformed normalized expression ratios.

Total RNA from tissue samples was extracted using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions, digested by DNAse I and reverse-transcribed using hexameric random primers. Quantification of gene expression was performed by quantitative real-time PCR using the primers and probes reported in the Supplementary Table 3. The murine housekeeping gene GAPDH was used to normalize the results. All the amplifications were performed using a BIORAD CFX96 real-time machine, using pre-developed and custom-designed assays (Applied Biosystems, Foster City, CA, USA) or SYBRGreen (Bio-Rad Laboratories, Hercules, CA, USA). Quantification of individual vector DNA in muscle samples was performed by Q-PCR using SYBRGreen. A Taqman probe and primers that specifically recognize the CMV promoter, which is common to all the AAV vectors in the study, was used to quantify total viral genomes. Primers and probes for viral DNA quantifications are reported in Supplementary Table 3.