GSM9035780: Ms_3888_5ul_CRE_sgNC_5_60hFM_Rep2; Mus musculus; RNA-Seq
Total RNA was then isolated using QIAzol Lysis Reagent (QIAGEN) and extracted using the miRNeasy Mini Kit (Qaigen), with the inclusion of the on-column genomic DNA digestion step using the RNase-free DNase Kit (Qaigen), following the standard protocols. RNA was quantified using the NanoDrop 2000 Spectrophotometer (ThermoFisher Scientific), and 1 μg of total RNA was used for complementary DNA (cDNA) synthesis using the SuperScript III Reverse Transcriptase enzyme (Thermo Fisher Scientific) following the manufacturer's instructions. RiboErase RNA-seq libraries were prepared using 200–1,000 ng of total RNA extracted from mouse organoids. Ribosomal RNA was eliminated through enzymatic digestion of the specific probe-bound duplex rRNA using the KAPA RNA Hyper+RiboErase HMR kit (Roche). The remaining RNA was then fragmented to approximately 200-300 bp by heat treatment in fragmentation buffer. Following fragmentation, double-stranded cDNA was synthesized, and end-repair and ligation procedures were carried out using New England Biolabs (NEB) adapters. The final library preparation involved amplification using the 2x KAPA HiFi HotStart mix and NEB dual barcodes, following the manufacturer's protocol. Library quality was assessed using an Agilent 2100 Bioanalyzer to determine product size and concentration. Paired-end sequencing was performed on the Illumina HiSeq 2500 platform, with 2 × 100 nucleotide read lengths and sequence coverage of 15–20 million paired reads per sample.