GSM9237661: Cho-org-P0-M2; Mus musculus; RNA-Seq
Total RNA was isolated from organoids using the Direct-zol™ RNA MiniPrep Kit according to the manufacturer's protocol. 1ug total RNA used for sequencing. A total of 1 µg total RNA per sample was used as input material for the mRNA library preparation. Most of Eukaryote 'mRNA have polyA tails, which are enriched by Oligo (dT) magnetic beads. Then, the obtained mRNA was randomly interrupted by bivalent cations, and the first strand of cDNA was synthesized in the 1st Strand Enzymes Reverse transcriptase system using the segmented mRNA as the template and the random oligonucleotide as the primer, and the second strand of cDNA was synthesized using the 2nd Strand Enzymes and dNTPs. Purified double-stranded cDNA undergoes end repair, A-tailing, and ligation with sequencing adapters. cDNA with an insert size of approximately 200-300 bp is screened. PCR amplification is performed, and the PCR product is purified again, ultimately obtaining a library. And then, library quality was assessed on the Agilent 4200 TapeStation. RNA purity and quantification were evaluated using the NanoDrop 2000 spectrophotometer (Thermo Scientific, USA). RNA integrity was assessed using the Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). Then the libraries were constructed using cDNATruePrep DNA Library Prep Kit V2 for Illumina (vazyme, Nanjing, China) according to the manufacturer's instructions. The transcriptome sequencing and analysis were conducted by OE Biotech Co., Ltd. (Shanghai, China).