title: GSM8992212: WT4_Aligned.sortedByCoord.out.bam_WT; Mus musculus; RNA-Seq
library_construction_protocol: Total RNA was isolated from calvarial cells using a combination of TRI reagent (Molecular Research Center, Cincinnati, OH, USA) and RNeasy-mini columns, including on-column deoxyribonuclease I digestion (Qiagen, Germantown, MD USA). RNA integrity was assessed using a 4200 TapeStation system. Directional cDNA libraries for mRNA-sequencing were prepared using polyA-mRNA from individual biological replicates using NebNext Ultra reagents. One μg of total RNA was utilized for mRNA isolation using poly-A magnetic beads and was fragmented prior to first and second strand cDNA synthesis. Libraries were generated via PCR including indexed barcodes. Following purification with AmpureXP beads libraries were quantitated using Qubit dsDNA reagents. Pooled cDNA libraries were sequenced using a Nextseq 550 instrument. All libraries were prepared and sequenced in a single batch.