Instrument: Illumina HiSeq 2500
Strategy: RNA-Seq
Source: TRANSCRIPTOMIC
Selection: cDNA
Layout: SINGLE
Construction protocol: RNA was isolated from HEK 293T and IMR-90 cells using TRIzol extraction. Four biological replicates from each cell line were DNase treated, and Ribo-Zero rRNA removal (Ribo-Zero, Epicentre) was utilized for three of the four RNA samples, leaving a non-Ribo-Zero depleted sample for rRNA expression analysis. cDNA synthesis was performed by using oligo-dT as well as random hexamers. Actinomycin D was added to the reaction to prevent any possible amplification from contaminating genomic DNA. During second strand synthesis, a dU/VTP mix was used to create directional libraries. Before library preparation, cDNA samples were Covaris-fragmented to 300 bp fragments. The samples were then end-filled, 3’ terminal A-extended and ligated to pre-annealed TruSeq indexed Illumina adapters. Uracil-DNA-glycosylase (UDG) treatment preceded the PCR reaction to cleave the uridine-containing strand and therefore identify the orientation of each transcript. The samples were multiplexed and sequenced using 100 bp single-end read sequencing on the Illumina HiSeq 2500 in our institutional Epigenomics Shared Facility.