title: GSM5179350: KO2_RNA-seq; Mus musculus; RNA-Seq
library_construction_protocol: RNA isolation was performed using the TRIZOL reagent (Invitrogen) according to manufacturer's instructions. Tissue or cells were homogenized using a glass-Teflon in 1 ml or 500 µl TRIZOL reagent on ice and phase separation was achieved with 200 µl or 100 µl chloroform. After centrifugation at 12,000× g for 15 min at 4°C, RNA was precipitated by mixing aqueous phase with equal volumes of isopropyl alcohol and 0.5 µl 20 mg/ml glycogen. Precipitation were dissolved in DNase/RNase-free water (not diethylpyrocarbonate treated, Ambion). Total RNA was extracted as described above. The concentration and quality of RNA was measured with Nanodrop 2000c (Thermo Fisher Scientific) and an Agilent 2100 Bioanalyzer (Agilent Technologies), respectively. RNA-seq libraries were constructed by NEBNext Poly(A) mRNA Magnetic Isolation Module (NEB E7490) and NEBNext Ultra II Non-Directional RNA Second Strand Synthesis Module (NEB E6111). Briefly, mRNA was extracted by poly-A selected with magnetic beads with poly-T and transformed into cDNA by first and second strand synthesis. Newly synthesized cDNA was purified by AMPure XP beads (1:1) and eluted in 50 μl nucleotide-free water. RNA-seq libraries were sequenced by Illumina NovaSeq 6000 platform with pair-end reads of 150 bp. The sequencing depth was 60 million reads per library.
attributes.tag: GEO Accession
attributes.value: GSM5179350