GSM6944419: sample26_contralateral sensorimotor cortex, 24 hour; Rattus norvegicus; RNA-Seq
Total RNA was extracted from 100-150 mg of flash-frozen rat somatosensory cortex and striatum tissue. Glycoblue (Invitrogen #AM9515) was added to assist with RNA pellet isolation. Pellets were washed with 75% ethanol and allowed to dry before being dissolved in 20 μL of RNase-DNase free water. 2 μL of 10x DNase I Buffer (New England Biolabs, #m0303s), 1 μL of rDNase I (ThermoFisher Scientific, AM2235), and DNase Inactivation Reagent (ThermoFisher Scientific, AM1907) were added to each RNA sample for purification.  Library preparation was performed by the QB3-Berkeley Functional Genomics Laboratory at UC Berkeley. mRNA selection was done with the oligo-dT beads. Both total RNA and oligo dT bead selected mRNA quality was assessed on an Agilent 2100 Bioanalyzer. Libraries were prepared using the KAPA RNA Hyper Prep kit (Roche KK8540). Truncated universal stub adapters were ligated to cDNA fragments, which were then extended via PCR using unique dual indexing primers into full length Illumina adapters. Library quality was checked on an AATI (now Agilent) Fragment Analyzer. Libraries were then transferred to the QB3-Berkeley Vincent J. Coates Genomics Sequencing Laboratory, also at UC Berkeley. Library molarity was measured via quantitative PCR with the KAPA Library Quantification Kit (Roche KK4824) on a BioRad CFX Connect thermal cycler. Libraries were then pooled by molarity and sequenced on an Illumina NovaSeq 6000 S4 flowcell for 2 x 150 cycles, targeting at least 25M reads per sample.