GSM7789701: shRen rep5; Mus musculus; RNA-Seq
Mammary gland tissue was dissociated using gentle collagenase/hyaluronidase (Stemcell), mammary epithelial cells were further isolated using 0.25% trypsin (Gibco) and 5 mg/mL dispase (Merck). GFP+ mKate2+ shRNA-expressing luminal and basal cells were sorted via FACS using EpCAM and CD49f surface markers (antibodies 12-9326-42 from eBioscience and 313631 from Biolegend). RNA was extracted using Rneasy Micro Plus Kit (Qiagen). 600 ng of RNA was used for cDNA amplification using the NEB Kit #E6421 "NEBNext® Single Cell/Low Input cDNA Synthesis & Amplification" RNA-Seq libraries were generated using the NEBNext Ultra II RNA Library Prep Kit for Illumina (NEB #E7770/E7775) according to the manufacturer's protocol. 8 cycles were used for library amplification.