title: GSM8100720: con,biol rep 3,RNAseq; Mus musculus; RNA-Seq
library_construction_protocol: 5 cm of mouse jejunum tissue was excised and longitudinally opened, and resuspended in RPMI 1640 solution (10% FBS added) into gentleMACS C tubes. To digest the tissues, DNase I (DN25, Sigma-Aldrich) at 1:2000 dilution, Protease Inhibitor Cocktail (HY-K0010, MedChemExpress), Dispase II (40104ES80, Yeasen) and Liberase (5401054001, Roche) at 1:37.5 dilution were added. The tissue was then digested using a gentleMACS Dissociator (Miltenyi Biotec).Epithelial cells (CD45−CD11b−CD3−B220−EpCAM+) were collected using BD FACS Aria II SORP (BD Biosciences). After the RNA samples were tested, eukaryotic mRNA was enriched with magnetic beads with Oligo(dT) . Subsequently, mRNA was interrupted into short fragments by adding fragmentation buffer. One-stranded cDNA was synthesized by using six-base random primers (random hexamers) with mRNA as template, and then two-stranded cDNA was synthesized by adding buffer, dNTPs, and DNA polymerase I and RNase H. The purified double-stranded cDNA was first subjected to end repair, addition of A-tail and ligation of sequencing junctions, followed by fragment size selection with AMPure XP beads. Finally, PCR amplification was performed and the PCR products were purified by AMPure XP beads to obtain the final library. After the library was constructed, Qubit 2.0 was used to perform preliminary quantification and dilution of the library, followed by Agilent 2100 to detect the insert size of the library, and after the inserts met the expectation, Q-PCR was used to accurately quantify the effective concentration of the library to ensure the quality of the library.