title: RNA-Seq Data for HG03516 Replicate 2
design: Library preparation was performed by the Genome Technology Access Center (GTAC) at the McDonnell Genome Institute. Total RNA integrity was evaluated using an Agilent Bioanalyzer. Libraries were prepared using 500 ng to 1 g of total RNA. Ribosomal RNA removal was conducted via an RNase-H-based method, utilizing RiboErase (Kapa Biosystems). Following ribosomal RNA depletion, mRNA was fragmented by incubation in reverse transcriptase buffer and heating at 94C for eight minutes. This fragmented mRNA was reverse transcribed into cDNA using the SuperScript III Reverse Transcriptase enzyme (Life Technologies), in accordance with the manufacturer's protocols, and employing random hexamers. A second-strand cDNA synthesis was performed, converting the single-stranded cDNA into double-stranded cDNA (ds-cDNA). The ds-cDNA was then subjected to end repair processes to create blunt ends, followed by the addition of an adenine base to the 3' ends. Illumina sequencing adapters, which contained unique dual index tags, were ligated to these prepared ends. The adapter-ligated cDNA fragments underwent PCR amplification for 12 to 15 cycles using primers that included unique dual index tags. Sequencing was performed on an Illumina NovaSeq 6000 system, producing paired-end reads that extended 150 bases in length. Resulting sequencing data is unstranded.