GSM8986622: fibroblasts, aldosterone sample 2, biol rep 1; Mus musculus; RNA-Seq
Fibroblasts were lysed directly in the plates using 100 uL of TRIzol reagent (Invitrogen) and detached from the plates using a cell scraper. RNA was isolated using the Direct-zol RNA microprep kit (Zymo Research). 12 uL of total RNA from each sample at 50 ng/uL were sent to Novogene Co., Ltd. (Davis, CA) for sequencing.  RNA integrity was assessed using a Bioanalyzer 2100 system (Agilent  Technologies). Messenger RNA was purified from total RNA using poly-T oligo-attached magnetic beads. After fragmentation, the first strand cDNA was synthesized using random hexamer primers, followed by the second strand cDNA synthesis using either dUTP for directional library or dTTP for non-directional library. The nondirectional library was ready after end repair, A-tailing, adapter ligation, size selection, amplification, and purification. The directional library was ready after end repair, A-tailing, adapter ligation, size selection, USER enzyme digestion, amplification, and purification. The library was checked with Qubit and real-time PCR for quantification and bioanalyzer for size distribution detection.