Northernmax gly sample loading dye

Total RNA was isolated from fibroblasts or lymphoblasts using the TRIzol reagent (Invitrogen) and following manufacturer's instructions. Cells were lysed in the culture vessel by the addition of TRIzol and pipetting up and down multiple times. Cells grown in suspension were pelleted and the supernatant removed before lysis. Chloroform (0.2 ml) was added to lysates and shaken by hand for 15 s to mix, then incubated at room temperature for 3 min. Samples were centrifuged at 12 000 ×g 15 min at 4 °C, and the upper aqueous phase was transferred to a new tube. RNA was precipitated in 0.5 ml isopropanol, and rotated for 10 min at room temperature. After centrifugation, the supernatant was decanted, and the RNA pellet was washed with 1 ml 75% ethanol. The ethanol was removed, the pellet was air dried, and the RNA was resuspended in diethylpyrocarbonate-treated water and stored at − 80 °C. To prepare RNA for separation by gel electrophoresis, equal volumes of RNA and NorthernMax-Gly Sample Loading Dye (Ambion) were mixed and incubated at 50 °C for 30 min to eliminate secondary structures. RNA samples were then separated by gel electrophoresis in a 1% agarose BPTE gel (100 mM PIPES, 300 mM Bis-Tris, 10 mM EDTA pH 8.0) in 1 × BPTE buffer at 5 V per cm.

Total RNA (10 μg for 10- and 20-year-old rhesus macaque brain samples, 2 μg for fetal macaque hippocampal primary neurons) was denatured using NorthernMax^®-Gly sample loading dye (Ambion) and resolved on 1.2% agarose gel in MOPS buffer. The gel was soaked in 1 × TBE for 20 min and transferred to a Hybond-N + membrane (GE Healthcare) for 1 h (15 V) using a semi-dry blotting system (Bio-Rad). Membranes were dried and UV-crosslinked with 150 mJ/ cm² at 254 nm. Pre-hybridization was done at 68 °C for 1 h, and using DIG Northern Blot Starter KIT (Roche) DIG-labeled in vitro transcribed circCACNA2D1, circCACNA1E, CDRA1s, and circMbl junction site-targeting probes were hybridized overnight. The membranes were washed three times in 2 × SSC, 0.1% SDS at 68 °C for 30 min, followed by three 30 min washes in 0.2 × SSC, 0.1% SDS at 68 °C. The immunodetection was performed with anti-DIG AP-conjugated antibodies. Immunoreactive bands were visualized using CDP star reagent (Roche) and a LAS-4000 detection system (GE Healthcare).

Total RNA (10 μg for 10- and 20-year old male macaque brain samples, 2 μg for fetal macaque hippocampal primary neurons) was denatured using NorthernMax^®-Gly sample loading dye (Ambion) and resolved on 1.2% agarose gel in MOPS buffer. The gel was soaked in 1×TBE for 20 min and transferred to a Hybond-N+ membrane (GE Healthcare) for 1 h (15 V) using a semi-dry blotting system (Bio-Rad). Membranes were dried and UV-crosslinked with 150 mJ/cm² at 254 nm. Pre-hybridization was done at 68 °C for 1 h, and using the DIG Northern Blot Starter Kit (12039672910, Roche). DIG-labeled in vitro transcribed Gria1 junction site-targeting circular, and junction site-nontargeting linear probes were hybridized overnight. The membranes were washed three times in 2 × SSC, 0.1% SDS at 68 °C for 30 min, followed by three 30 min washes in 0.2 × SSC, 0.1% SDS at 68 °C. The immunodetection was performed with anti-DIG AP-conjugated antibodies. Immunoreactive bands were visualized using CDP star reagent (Roche) and a LAS-4000 detection system (GE Healthcare). The primer sequences of probes were seen in Supplementary Data 3.