title: GSM4773898: 4_2_box1; Rattus norvegicus; RNA-Seq
library_construction_protocol: Total RNA was isolated from 30 μl of lysate using TRIzol LS reagent (Life technologies, 10296028). Lysate volume was adjusted to 100 μl and 300 μl Trizol LS (3X volume) reagent was added. Then sample was incubated for 5 min at room temperature and 80 μl (1/5 volume) of chloroform was added following by shaking for 15 sec, incubation for 10 min room temperature and centrifugation for 15 min at 14,000 × rpm at 4°C. The aqueous phase containing the RNA was transferred to a new tube, 200 μl (1X volume) of isopropanol (VWR, 87000–048) was added to the aqueous phase and mixed well by inverting several times following by incubation for 10 min at room temperature and centrifugation for 25 min at 14,000 × rpm at 4°C. Total RNA precipitate formed a white gel-like pellet at the bottom of the tube. The pellet was washed with 1 ml of 75% ethanol and centrifuged for 5 min at 7500 × rpm at 4°C. The pellet was washed one more time in 1 ml of 100% ethanol and centrifuged again at the same settings. The RNA pellet was dried for 10 min at room temperature and resuspended in 30 μl of RNase-free water. The RNA sample was cleaned with RNeasy MinElute Cleanup Kit (Qiagen, 74204). Total RNAs extracted from each sample were sent to University of Minnesota Genomic Center for library preparation and RNA-seq with standard QC metrics (RNA quantity > 1 µg, RIN > 9.0, OD260/280 > 1.9 ), NovaSeq S2 2x50bp, > 32M PE reads per library.