Dynabeads mrna purification kit

Total RNA was isolated from MOLM13 cells using the RNeasy midi kit (Quiagen) and polyA+ RNA was purified from 30 μg total RNA using the Dynabeads mRNA Purification Kit (Thermo Fisher Scientific) according to the manufacturer’s instructions. For production of unmodified 7SK RNA, synthetic double stranded DNA template for in vitro transcription (IVT) was produced by hybridization of synthetic Megamer® Single-Stranded DNA Fragments (IDT) containing the 7SK sequence downstream of a T7 promoter (Table S3). 500ng of double stranded DNA template were used in 20 μl IVT reactions for 1h using the TranscriptAid T7 High Yield Transcription Kit (Thermo Fisher Scientific), following the manufacturer’s instructions. The RNA product was purified using the RNA Clean & Concentrator kit (Zymo Research). Wild Type and ime4Δ yeast cells were collected after 4h in sporulation medium and total RNA was extracted with acid phenol:chloroform:isoamyl alcohol as previously described^{47 (link)}. polyA+ RNA was purified from total RNA using the Dynabeads mRNA Purification Kit (Thermo Fisher Scientific) as above.

mRNA samples were prepared in three biological replicates. Cells were grown in 15 cm dishes to 70–80% confluency. Cell pellets were resuspended in 1.5 mL TRI reagent (Sigma). 300 µL Chloroform (Applichem) was added, and samples were mixed and centrifuged at 4 °C, max. speed for 15 min. The upper phase was transferred to a fresh tube and subjected to isopropanol precipitation. Totally, 120 µg of RNA were treated with 100 U DNase I (Roche) at 37 °C for 30 min and purified by phenol-Chloroform extraction and Ethanol precipitation. 100 µg total RNA was used as an input for mRNA enrichment. Polyadenylated RNA was isolated using Dynabeads mRNA Purification Kit (Invitrogen) according to the manufacturer’s instructions. rRNA was depleted using NEBNext rRNA depletion kit v2 (Human/Mouse/Rat) (New England Biolabs) according to the manufacturer’s instructions, followed by another round of mRNA enrichment using Dynabeads mRNA Purification Kit (Invitrogen). The absence of rRNA in the final mRNA preparations was confirmed by capillary electrophoresis on a Fragment Analyzer (Supplementary Fig. 14).

Whole-transcriptome analysis was performed using pooled brain tissues of SN of mice with MPTP-CBE-induced PD with GCase dysfunction, MPTP-induced PD, CBE and control mice (vehicle) with NaCl. For this purpose, 5 mg of brain tissue was taken from each of the four animals in each group. Total RNA was extracted from brain tissue with Trisol reagent and PureLink RNA micro Kit (PureLink RNA micro Kit, Invitrogen, CA, USA), according to the manufacturer’s instruction. The quality was checked with a BioAnalyser (2100 Bioanalyzer Instrument, Agilent, CA, USA) and RNA 6000 Nano Kit (RNA 6000 Nano Kit, Agilent, CA, USA). PolyA RNA was purified with Dynabeads^® mRNA Purification Kit (Dynabeads^® mRNA Purification Kit, Ambion, TX, USA). The Illumina library was made from polyA NEBNext^® Ultra™ II RNA Library Prep (NEBNext^® Ultra™ II RNA Library Prep, NEB, MA, USA), according to the manual. Sequencing was performed on a HiSeq1500 (Illumina, CA, USA) with 50 bp read length. At least ten million reads were generated for each sample.