Total RNA was extracted with TRIzol reagent (Invitrogen) according to the protocol supplied by the manufacturer and a previous report (Jin et al., 2016 (link)). RNA purity and concentration were determined by NanoDrop ND-1000 Spectrophotometer (Nano Drop Technologies, USA) using absorbance at 260 nm and 280 nm. cDNA was synthetized using the Qiagen Whole Transcriptome Kit (Qiagen, USA) and analyzed by real-time PCR using the following primer sets: NELL2 sense primer, 5′-GGT AGC CGT CTC TGC ACT CA-3′, NELL2 antisense primer, 5′-TGT AGA AAC GGA GCG TG-3′; Robo2 sense primer, 5′-GCT ACG ATC CAA GAC CAA GG -3′, Robo2 antisense primer, 5′-CAG ACT CTG TCA CAT CCA GC-3′; Robo3 sense primer, 5′-GGA GAT CCC CAG CCC AAT CT-3′, Robo3 antisense primer, 5′-TCG GCC CAC ACT GTT TTC T-3′; GAPDH sense primer, 5′-GGG GCC AAA AGG GTC ATC AT-3′, GAPDH antisense primer, 5′-GTG ATG GCA TGG ACT GTG GT-3′. Real-time PCR experiments were performed by BrightGreen 2× qPCR MasterMix-ROX (ABM, Canada) using the StepOnePlus Real-Time PCR System (Applied Biosystems; Thermo Scientific) for ~40 cycles. Data were normalized for gene expression by using GAPDH as an internal control. The 2–ΔΔCT method was used to analyze the relative quantification of gene expression.