GSM8821302: Mouse embryonic stem cells (mESCs) in 2iLIF ground state: E14_2iLIF; Mus musculus; RNA-Seq
Total RNAwas extracted from mESCs in 2iLIF and cEpiSC after conversion by RNeasy® Plus Mini Kit (Qiagen) 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 with the fragmented RNA as template. To synthesize the second-strand cDNA, the synthesis reaction system is prepared and dUTP is used to replace dTTP. After obtaining the double strand cDNA product, it is converted to blunt end with end repair 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 library adapters are ligated to the two ends of the cDNA with ligation reaction. 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 circularized DNA products. Any 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 sequencing reads with PE 100/150 bases length are generated on G400 platform (BGI-Shenzhen, China).