GSM6657709: ACH-2  No drug control - replicate 2; Homo sapiens; miRNA-Seq
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. NEBNext small RNA sample library preparation kit (NEB; Ipswich, MA) was used to prepare the library with a modified approach for precise miRNA library size selection that allowed the kit to process low input and low-quality RNA with higher library recovery and miRNA read alignment.  After 15 cycles of PCR with 100 ng total RNA as input, the libraries with unique indices were pooled, column cleaned, and combined with a custom-designed DNA ladder containing 135 and 146 base pairs (bp) purified PCR amplicon.  This size range corresponds to a 16-27 nt insert miRNA library that covers all miRNAs.  Agarose gel electrophoresis and gel excision were carried out.  The library pool ranging from 135 to 146 bp, including the DNA marker, was gel purified and quantified using the NEBNext Library Quant kit (NEB) in the QuantStudio 5 Real-Time PCR System (Thermo Fisher Scientific; Waltham, MA).  The first round of sequencing was performed using an Illumina Nextseq 550 sequencer to generate a few million reads to measure the relative concentration of each library.  For the final data analysis, the capacity of each library was adjusted to generate the predicted number of equal reads from each sample.