title: GSM6659007: ACH-2 with fentanyl replicate 2; Homo sapiens; RNA-Seq
library_construction_protocol: Total RNA of fentanyl-treated and untreated ACH-2 cells was isolated using the commercially available miRNA Isolation Kit (miVana; Applied Biosystems; Carlsbad, CA) according to the manufacturer's protocol.  A NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific; Waltham, MA) was used to determine total RNA concentration and purity.  A 2100 Bioanalyzer (Agilent; Santa Clara, CA) and agarose electrophoresis were used to assess RNA integrity. Bioanalyzer was used to test the quality of total RNA (Agilent; Santa Clara, CA).  The Poly(A) mRNA Magnetic Isolation Module (NEBNext; Ipswich, MA) was used to isolate polyA RNA for library preparation with 1 µg of high-quality total RNA as input.  SMARTer Apollo automated NGS library prep system (Takara Bio USA; Mountain View, CA) was used for enrichment of polyA RNA.  For library preparation, the New England BioLabs NEBNext Ultra II Directional RNA Library Prep kit (PCR cycle number 8) was used.  The individual indexed libraries were proportionally pooled, and sequenced on the NextSeq 550 sequencer (Illumina; San Diego, CA) under the sequencing setting of single read 1x85 bp after QC and quantification via real-time qPCR (NEBNext Library Quant Kit; New England BioLabs; Ipswich, MA).