GSM3004225: RUES-1 hESC OGT-WT; Homo sapiens; RNA-Seq
Total RNA was isolated from cells harvested from confluent 60 mm dishes using the RNeasy Plus Mini Kit (Qiagen) according to the manufacturer's instructions. The concentration and integrity of the extracted total RNA were estimated using the Qubit® 2.0 Fluorometer (Invitrogen) and Agilent 2100 Bioanalyzer (Applied Biosystems, Carlsbad, CA, USA), respectively. Five hundred nanograms of total RNA was required for downstream RNA-seq applications. Polyadenylated RNAs were isolated using NEBNext Magnetic Oligo d(T)25 Beads. Next, first strand synthesis was performed using NEBNext RNA first strand synthesis module (New England BioLabs Inc., Ipswich, MA, USA). Immediately, directional second strand synthesis was performed using NEBNExt Ultra Directional second strand synthesis kit. The NEBNext® DNA Library Prep Master Mix Set for Illumina® was then used to prepare individually bar-coded next-generation sequencing expression libraries as per manufacturer's recommended protocol. Library quality was assessed using the Qubit 2.0 Fluorometer, and the library concentration was estimated by utilizing a DNA 1000 Chip on an Agilent 2100 Bioanalyzer. Accurate quantification for sequencing applications was determined using the qPCR-based KAPA Biosystems Library Quantification Kit (Kapa Biosystems, Inc., Woburn, MA, USA). Paired-end sequencing (50 bp) was performed on an Illumina HiSeq2500 sequencer to obtain approximately 50 million reads per sample. Raw reads were de-multiplexed using bcl2fastq Conversion Software (Illumina, Inc.) with default settings.
GEO Accession
GSM3004225