GSM8946629: Young_Male_3_DAT; Mus musculus; RNA-Seq
Mice were anesthetized with isoflurane and rapidly decapitated. To minimize activity induced transcriptomic changes, the brain was immediately removed and placed in ice-cold choline chloride cutting solution. 600 µm-thick horizontal brain slices were collected, accounting for the entire rostral-caudal length of the ventral midbrain. Sections were moved onto a pre-chilled collection block, where the surrounding tissue (such as the pons, hippocampus, and cortex) was removed. Remaining bilateral midbrain portions (containing the substantia nigra pars compacta [SNc] and the ventral tegmental area [VTA]) were collected into Eppendorf tubes and flash-frozen for RNA preservation. All samples were stored in -80oC freezer until the time of TRAP and mRNA isolation. midbrain slices were placed in 200 µl of ice-cold homogenization buffer (50mM Tris, pH 7.4; 12mM MgCl2; 100mM KCl; 1% NP-40; 1 mg/ml sodium heparin; 1mM DTT) supplemented with 100 μg/mL cycloheximide (Millipore, cat# C4859–1ML), 200 units/ml RNaseOUT Recombinant Ribonuclease Inhibitor (ThermoFisher, cat# 10777019), and 1× cOmplete, EDTA-free Protease Inhibitor Cocktail (Milipore, cat# 11836170001). Homogenization used a cordless motor pestle (Kimble #749520-0090 and #749540). After initial homogenization, an additional 500 µl of buffer was added to the samples, washing the pestle in between pulses. Homogenate solution volume was then brought to a total of 1.5 ml and centrifuged at 12,000 g for 10 min at 4oC. After centrifugation, 100 µl of the supernatant was removed and set aside on ice (“input fraction”). The remaining supernatant (~900 µl of “positive fraction”) was transferred into a fresh tube and incubated with 1 µl anti-GFP antibody (Abcam, cat# ab290). Both input and positive samples were placed in an end-over-end rotating mixer and incubated for 1 h at 4oC. Pre-washed Dynabeads protein G (Invitrogen, cat# 10003D; 30 µl per sample) were then added to the positive fraction and the final solution incubated overnight (~16 h) in a rotating mixer at 4oC. Positive tubes were then placed in a DynaMag-2 magnet and supernatant removed and saved as the “negative fraction”. The magnetic beads bound with GFP-labelled polyribosomes/mRNAs, were washed with high-salt buffer (50mM Tris, pH 7.5; 12mM MgCl2; 300mM KCl; 1% NP-40; 100 μg/ml cycloheximide; 2mMDTT) three times. After the final wash, Dynabeads were separated from the GFP-labelled polyribosomes/mRNAs by adding 350 µl of Buffer RLT (Qiagen) and 3.5 µl of 2-β mercaptoethanol, proceeding with incubation in a benchtop ThermoMixer (Eppendorf) for 10 min at 22oC. The eluted solution (“positive fraction”), free of magnetic beads, was placed in a fresh tube. mRNA isolation was carried out using RNeasy Mini kit (#74104, QIAGEN), following manufacturer's suggested protocol. Isolated mRNA was then quantified with a Nanodrop spectrophotometer (ThermoFisher, model# ND-ONEC-W) and its quality measured in a 4150 Tapestation analyzer (Agilent, model# G2992AA) using HSRNA ScreenTape (Agilent, cat# 5067-5579). Directional mRNA libraries were prepared using NEBNext Ultra II Kit for Illumina (NEB, cat# E7760L) in accordance to the manufacturer's directions, and following previously established protocol (Chucair-Elliott et al., 2020; Ocanas et al., 2022). In summary, each sample of both positive and input fractions (ranging 3.5-35 ng of mRNA) were individually captured by poly-A RNA using NEBNext Poly(A) mRNA Magnetic Isolation Module (NEB, cat# NEBE7490L). mRNA was then eluted from oligo-dT beads, fragmented using a thermal cycler at 94°C for 15 min, and first and second strands of cDNA were individually synthesized following manufacturer's guidance (NEB, cat# E7760L). The final double-stranded cDNA was purified with the use of SPRISelect Beads (Beckman Coulter, cat# B23318) and eluted in 50 µl 0.1x TE buffer. Next, adaptor ligation was performed using 100-fold dilution of the NEBNext adaptor in dilution buffer (NEB, cat# E6609L), and PCR of the ligated products was conducted using the NEBNext Ultra II Q5 Master Mix (NEB, cat#7760L) and unique index primers (NEB, cat# E6609L), both provided with aforementioned kits, in accordance to manufacture's protocol (14 cycles). Purified libraries were then quantified using HS dsDNA Qubit kit (ThermoFisher, cat# Q33230). Library integrity, verified in a 4150 Tapestation analyzer (Agilent, model# G2992AA) using HS D1000 ScreenTape (Agilent, cat# 5067-5584), revealed an average peak size of 332bp. Libraries for each sample were then pooled at 5 nM concentration and sequenced by the OMRF[Author's institution] Clinical Genomics Center using the Illumina NovaSeq 6000 system (S4 PE150).