title: GSM9382827: Ctrl2; Mus musculus; RNA-Seq
library_construction_protocol: Total RNA was isolated using the Trizol Reagent (Invitrogen Life Technologies), after the total RNA was qualified and quantified as follows: (1) RNA purity and concentration were then examined using NanoDrop spectrophotometer (Thermo Scientific) and Qubit 4.0; (2) RNA integrity and quantity were measured using the Agilent 2100/4200 system. Three micrograms of RNA were used as input material for the RNA sample preparations. Sequencing libraries were generated according to the following steps. Firstly, mRNA was purified from total RNA using poly-T oligo-attached magnetic beads. Fragmentation was carried out using divalent cations under elevated temperature in fragmentation buffer. First strand strand was synthesized by random hexamers, and the second cDNA strand was synthesized by adding buffer, dNTPs, RNase H and DNA polymerase I. Double stranded cDNA was repaired and A was added to the 3 'end. Hieff NGS® DNA Selection Beads were used for purification and fragment Selection. After purification and fragment selection, the products were amplified and enriched by PCR. Qubit was used for quantitative . The target region library of the duplex was denatured, cycled, and digested to yield a single-stranded circular DNA. Single-stranded circular DNA is amplified by a rolling circle amplification (RCA), known as DNA nano balls (DNB). After library construction, Qubit was used for quantitative.