GSM5752801: 2134_Fetal_GD21 [SP0224-F06]; Rattus norvegicus; RNA-Seq
Subsamples of liver tissue (∼30–50 mg) were collected from NBP2-exposed dams (GD18 and GD21) and fetuses (GD18 and GD21) were placed in polypropylene microcentrifuge tubes containing 500 µL TRIzol Reagent (Invitrogen, Carlsbad, CA, USA) on ice, then individually homogenized using a Bullet Blender (Next Advance, Troy, NY, USA) with 1 mm zirconium oxide beads and stored at −80 °C prior to RNA extraction. RNA extraction was conducted according to TRIzol Reagent manufacturer specifications using chloroform and isopropanol. Following extraction, RNA was purified using RNeasy Mini Kit (Cat no. 74104; Qiagen, Hilden, Germany). RNA concentration and purity (260:280 ratio ≥ 1.8) were determined with a NanoDrop 2000 spectrophotometer (Thermo Scientific, Waltham, MA, USA). Libraries were prepared as described in Trejo et al. (2019) (https://doi.org/10.1371/journal.pone.0212031). Purified RNA was processed in annealing buffer with detector oligos (DOs), probes that have high selectivity for 21,119 protein coding genes in the rat genome (Rat Whole Transcriptome Assay on the Templated Oligo Detection Assay platform; TempO-Seq, BioSpyder Technologies, Carlsbad, CA, USA). After oligo ligation, a nuclease mixture was added to digest unbound or erroneously bound DOs. A final ligase mix was added to facilitate addition of a single primer pair. The ligases were deactivated with heat and the probes were amplified by PCR, which added sample specific barcodes that flank the target sequence and are inserted into the standard Illumina adaptors. PCR-amplified and barcoded samples were then pooled into a single library, purified and sequenced. TempO-Seq RNA samples were sequenced on the Illumina HiSeq 2500  platform with 50-bp single-end reads to a target read depth of 5 million. Overly abundant transcripts were attenuated equally across all samples to save sequencing reads.