GSM8513061: Microglia, TDP-43_Q331K, 3Months, rep2; Mus musculus; RNA-Seq
To obtain a high RNA quality and yield with small cell amounts, we used the Single Cell RNA Purification Kit (Norgen Biotek Corporation, Thorold, ON, Canada) according to the manufacturer's instructions. RNA was quantified with Qubit fluorometer (Thermo Fisher Scientific, Inc.). Finally, RNA quality was assessed with Agilent 2100 Bioanalyzer HS for RNA. Omomyc: After 14 days, primary astrocytes were replated (4 × 10⁵ cells/well), treated the next day with 40 μM Omomyc for 48 h, and harvested for RNA extraction (Total RNA Purification Kit, Norgen Biotek). Astrocytes and oligodendrocytes: Poly(A) RNA-seq library prep was performed according to the manufacturer's protocol (Lexogen CORALL mRNA-Seq V2 Library Prep Kits with UDIs cat. N. 177-182), adding Unique Molecular Identifier during the linker ligation step. Microglia: Poly(A) RNA-seq Lexogen QuantSeq 3' mRNA-Seq Library Prep Kit for Illumina. For Omomyc-treated and control cells, 1000ng of total RNA was used for Poly(A) enrichment and RNA-seq library preparation according to the Watchmaker mRNA Library Prep Kit protocol. For library construction, the Twist UMI Adapter System (Twist Bioscience) was included in the protocol. Final sequencing libraries were quantified with Qubit DNA HS Kit, analyzed on a Perkin Elmer LabChip GXII, pooled in equimolar amounts, and sequenced on a lane of SP Novaseq6000 flow cell, obtaining an average of 40 million PE150 reads (clusters) per sample.