GSM5829008: RNA-seq - siTfam - rep1; Mus musculus; RNA-Seq
OG2 mESCs samples, cells were lysed by 1 ml TRI Reagent, followed by adding 200 μl chloroform with robust vortex for 15 s. The mixture was centrifuged at 12,000 g at 4ºC for 15 min, than the RNA was precipitated with isopropanol at -20ºC for 30 min or overnight. And to facilitate precipitation, glycogen (Roche) was added if necessary. Total RNA, EpiTM mini longRNA-seq kit was used for library preparation. In brief, 10 μl IP RNA and 50 ng input RNA were used for library construction, total RNAs were digested by DNase I to remove residual DNAs, and RNAs were fragmented. Then the first cDNA was synthesized based on SMART (Switching Mechanism At the 5' end of RNA Transcript technology). OG2 mESCs experiments, followed by two-round of PCR amplifications and purification, the resultant cDNA library was evaluated using Bioptic Qsep100 Analyzer, and diluted to 500 pM for cluster generation and high-throughput sequencing (2 ×150 bp) on NovaSeq 6000 (Illumina).