RNA-Seq of HEK293T: Syn1 + aSyn
Purification of mCherry-tagged ectopically expressed condensates was obtained by adapting a protocol for P-body purification from Hubstenberger et al., 2017. For each expressing plasmid system in HEK293T (condition 1: 6xHis-TEV-mCherry-PP-hsSYN1 pCMV SYPH-FL; and condition 2 condensates: 6xHis-TEV-mCherry-PP-hsSYN1 + pCMV SYPH-FL + pCMV hsSNCA) cells were grown 90-95% confluence on 10 cm Petri dish prior to harvesting. Transfection of plasmids for condensate purification was performed at 80% of cell confluency using LipofectamineTM 2000 (InvitrogenTM, 11668027) 20 hours prior to harvesting. Upon application of condensate purification protocol (see Rankovic, Geisterfer et al., Methods for details) condensate enriched fraction was sorted on BD FACSAria III Fusion system (Neutral Density filters: 1.0; Nozzle: 70 m and Psi: 50). Sorted condensates were subjected to TRIzol based total RNA extraction. Library preparation and Illumina sequencing were done at the Genomics Platform Max-Delbrck-Centrum fr Molekulare Medizin | BIMSB (Berlin, Germany). For the SV-like condensate RNA analysis, cDNA libraries were prepared from total RNA followed by ribosomal RNA depletion and library amplification using SMARTer Stranded Total RNA-Seq Kit v3 Pico Input from Takara Bio USA, Inc. The libraries were multiplexed, and sequenced with paired end 100 bp read using SP Flowcell of NovaSeq 6000 in PE100 mode, Illumina. 50 million reads per sample passed the Illumina filters.
SUB15005565
B2