title: GSM6975335: D2 primary oviductal muscle cell WT2; Mus musculus; RNA-Seq
library_construction_protocol: Oviduct were isolated and cut into small pieces (1-2 mm) and incubated (30-45 min) in rotation at 37 ℃ in pre-warmed DMEM/F12 (1:1) (Gibco), 12 mM HEPES (Gibco), 1% penicillin/streptomycin (Gibco), 2 mg/ml trypsin (Sigma), 400 U/ml collagenase type I (Invitrogen), and 5 U/ml dispase (Invitrogen). The digested cells were passed through a 70-mm filter to obtain a homogenous population of smooth muscle cells. Cells were plated in 100 mm dishes, containing DMEM and Ham F-12 media (1:1) (Gibco) with 12% charcoal-stripped fetal bovine serum (FBS) and antibiotics (penicillin-streptomycin, Thermo Fisher, 100 µg/mL). After 4 h, the medium was replaced with DMEM/F-12 (1:1) containing 10% FBS. 2 h later, the cells were gently washed twice with PBS and samples were prepared for RNA-seq. total RNA was isolated using a RNeasy Micro Kit. The libraries were constructed by using a Universal RNA-Seq Library Preparation Kit (TECAN) per the manufacturer's protocol in which double-strand cDNA was generated using a mixture of random and poly(T) priming, followed by fragmentation of double-stranded cDNA, end repair, adaptor ligation, strand selection, targeted transcript depletion with AnyDeplete, and PCR amplification to produce the final library.