title: GSM7674516: midbrain_day_5_replicate_2; Mus musculus; RNA-Seq
library_construction_protocol: RNA was extracted using the TRIzol-chloroform based method. Tissue was lysed in TRIzol (ThermoFisher 15596018), and lysate was mixed with phenol:choloroform:isoamyl alcohol (ThermoFisher 15593049) in a 5:1 ratio. Protein and nucleic acids were separated into phases via centrifugation. The top aqueous layer containing the RNA was passed through a QIAshredder column (Qiagen 79656). Eluent was mixed 1:1 with 70% ethanol and applied to an RNease Mini column (Qiagen 74104). After a brief centrifugation, columns were washed once with RW1 buffer and twice with RPE buffer, as per manufacturer's recommendations. RNA was eluted in nuclease-free water. 1µg of total RNA in a concentration of 20ng/µL was shipped to LC Sciences (Houston, TX) for library preparation and RNA sequencing. Total RNA was enriched for mRNA, fragmented, and reverse transcribed into complementary DNA (cDNA). Second-stranded DNA was synthesized, A-bases were added to the blunt ends of either strand, and DNA was ligated to dual-index adapters. Size selection was performed and DNA was treated with a heat-labile UDG enzyme. Ligated products were amplified with PCR before sequencing.