title: GSM8162806: Dam 43, IAV-X31, E16.5 fetal whole brains; Mus musculus; RNA-Seq
library_construction_protocol: RNA from pooled E16.5 fetal whole brains was extracted using the RNeasy Mini Kit (Qiagen). Construction of the strand-specific RNAseq libraries and sequencing on the Illumina NovaSeq 6000 were performed at the Roy J. Carver Biotechnology Center at the University of Illinois at Urbana-Champaign.Purified DNAsed total RNAs were run on a Fragment Analyzer (Agilent) to evaluate RNA integrity. The total RNAswere converted into individually barcoded polyadenylated mRNAseq libraries with theKapa HyperPrep mRNA kit (Roche). Libraries were barcoded with Unique Dual Indexes (UDI's) which have been developed to prevent index switching.The adaptor-ligated double-stranded cDNAs were amplified by PCR for 10 cycles with the Kapa HiFi polymerase (Roche). The final libraries were quantitated with Qubit (ThermoFisher) and the average cDNA fragment sizes were determined on a Fragment Analyzer. The libraries were diluted to 10nM and further quantitated by qPCR on a CFX Connect Real-Time qPCR system (Biorad) for accurate pooling of barcoded libraries and maximization of number of clusters in the flowcell.