title: GSM9496435: PSC_2; Mus musculus; RNA-Seq
library_construction_protocol: Total RNA was purified using the RNeasy Kit (Qiagen; 74004). Beta-mercaptoethanol (ß-ME) was used as the reducing agent, and the RNA extraction was performed as described by the Qiagen procedure. The RNA concentration and integrity were verified with a bioanalyzer, and 50 million read-pairs per library were prepared and sequenced by the Massively Parallel Sequencing Shared Resource at OHSU. RNA quality was assessed using the Agilent 2100 Bioanalyzer. Libraries were prepared using the TruSeq Stranded mRNA kit (Illumina). Briefly, 200ng of total RNA was converted to cDNA using random hexamers. Synthesis of the second strand was done with the addition of dUTP, which enforced the stranded orientation of the libraries by blocking amplification off the second strand during the first round of PCR. Amplified libraries were profiled on the 4200 TapeStation (Agilent). Libraries were quantified for sequencing using an NGS Library Quantification Kit (Roche/Kapa Biosystems) on a StepOnePlus Real Time PCR Workstation (Thermo/ABI). Sequencing was done on a NovaSeq 6000 (Illumina). Base calling was done by RTA v3.4.4. Fastq files were assembled using bcl2fastq v2.20.0.422 (Illumina).