GSM9149286: DRG, Tmem45b-flox mice, RNA-seq, rep6; Mus musculus; RNA-Seq
Total RNA was extracted using TRIzol reagent (Thermo Fisher Scientific, Carlsbad, CA, USA), following the manufacturer's protocol. Library preparation is performed using Optimal Dual-mode mRNA Library Prep Kit (BGI-Shenzhen, China). A certain amount of RNA are denatured at suitable temperature to open the secondary structure, and mRNA is enriched by oligo (dT) attached magnetic beads. After reacting at a suitable temperature for a fixed time period, RNAs are fragmented with fragmentation reagents. Then First-strand cDNA is generated using random hexamer-primed reverse transcription, followed by a second-strand cDNA synthesis. The synthesized double strand cDNA is subject to end repairment reaction. After cDNA end repairment, a single 'A' nucleotide is added to the 3' ends of the blunt fragments through A tailing reaction. Then the reaction system for adaptor ligation configured to ligate adaptors with the cDNAs, and finally, the library products are amplified through PCR reaction and subjected to quality control. Next, the single-stranded library products are produced via denaturation. The reaction system for circularization is set up to get the single-stranded cyclized DNA products. Any uncyclized single stranded linear DNA molecules will be digested. The final single strand circularized library is amplified with phi29 and rolling circle amplification (RCA) to make DNA nanoball (DNB) which carries more than 300 copies of the initial single stranded circularized library molecule. The DNBs are loaded into the patterned nanoarray and PE150 bases reads are generated on T7 platform (BGI-Shenzhen, China).