NANOG 5 week left #3
The global gene expression profiles were characterized by next generation RNA sequencing using Illumina platform. To this end, total RNA was isolated for all conditions using RNeasy Mini Kit and quality control analysis was performed by RNA gel and Agilent Fragment Analyzer. Sequencing libraries were prepared as per standard Illumina protocols (Illumina Stranded Total RNA Prep with Ribo-Zero Plus), quality checked, and quantified by Kapa Biosystems qPCR. The multiplexed libraries were sequenced in pair-end (2 X 50 bp) on the NovaSeq 6000 at 300 pM with 1% loading control. Sequencing reads passed quality filter from Illumina RTA were first processed using FASTQC (v0.10.1) for sequencing base quality control. Then sample reads were aligned to the human reference genome (GRCm38) and GENCODE (version 22) annotation database using STAR2 [115]. Second round of QC using RSeQC [116] was applied to mapped bam files to identify potential RNASeq library preparation problem. Gene level raw counts were obtained using Subread [117] package.