title: GSM8877047: HepG2 cells, BPA, rep 4; Homo sapiens; RNA-Seq
library_construction_protocol: Total RNA was extracted from HepG2 cells using the NucleoSpin RNA kit (Macherey-Nagel, 740955) according to the manufacturer's. 2 μg of total RNA was submitted to the North Carolina State University Genomic Sciences Laboratory (Raleigh, NC, USA) for library construction and sequencing. Total RNA was assessed for quality using an Agilent 2100 Bioanalyzer with an RNA 6000 Nano Chip (Agilent Technologies, USA). Purification of messenger RNA (mRNA) was performed using the oligo-dT beads provided in the NEBNext Poly(A) mRNA Magnetic Isolation Module (New England Biolabs, USA). Complementary DNA (cDNA) libraries were constructed using the NEBNext Ultra Directional RNA Library Prep Kit (NEB) and NEBNext Multiplex Oligos for Illumina (NEB) using the manufacturer-specified protocol. Briefly, the mRNA was chemically fragmented and primed with random oligos for first strand cDNA synthesis. Second strand cDNA synthesis was then carried out with dUTPs to preserve strand orientation information. The double-stranded cDNA was purified, end repaired and “a-tailed” for adaptor ligation. Following ligation, samples were selected with a final library size (adapters included) of 350-550 bp using sequential AMPure XP bead isolation (Beckman Coulter, USA). Library enrichment was performed and specific indexes for each sample were added during the protocol-specified PCR amplification. The amplified library fragments were purified and checked for quality and final concentration using an Agilent 2200 Tapestation with a High Sensitivity DNA chip (Agilent Technologies, USA) and a Qubit fluorometer (ThermoFisher, USA). The final quantified libraries were pooled in equimolar amounts for clustering and sequencing on an Illumina NovaSeq 6000 DNA sequencer, utilizing a 150x2 bp paired end sequencing reagent kit (Illumina, USA). The software package Real Time Analysis (RTA) was used to generate raw base call files (bcl), which were then de-multiplexed by sample into fastq files.
identifiers.namespace: GEO
identifiers.id: GSM8877047_r1