title: GSM6234253: RNA_amWT-F_rep3; Mus musculus; RNA-Seq
library_construction_protocol: Ventricular tissue from 8-week mice was snap-frozen and cryoground. Bulk RNA was extracted using the Zymo Quick-RNA Miniprep Plus Kit (R1057), following the manufacturer's protocol with some modifications in the tissue digestion stage. All centrifugation steps were performed at 16,000 RCF at 4°C. 20 mg of cryoground tissue was mechanically homogenised on ice with disposable probes (Qiagen TissueRuptor II) in 350 µL of DNA/RNA Shield. 45 µL of Proteinase K mix was added to 300 µL of sample, incubated for 30 mins at 55°C, and centrifuged for 2 mins after incubation. The supernatant was transferred to new microcentrifuge tubes and 300 µL RNA Lysis Buffer was added at 1:1 to the supernatant. Thereafter, the RNA purification procedure followed the manufacturer's protocol, with some differences: half volume ethanol (300 µL) was added, in order to isolate RNAs ≥ 200 nt for poly(A) enrichment; DNase I treatment was performed in-column; RNA was eluted in non-DEPC-treated nuclease-free water. PolyA RNA was purified using the NEBNext Poly(A) mRNA magnetic isolation module (E7490) and DNA libraries were prepared using the NEBNext Ultra II Directional RNA library preparation kit for Illumina (E7765).