GSM5528223: C3TAG DCIS containing whole mammary gland 2 for scRNAseq; Mus musculus; RNA-Seq
For scRNAseq, whole mammary glands and mammary tumors were placed in 10 ml of a digestion medium containing EpiCultTM-B Mouse Medium Kit (#05610, StemCell Technologies), Col-lagenase/Hyaluronidase (#07912, StemCell Technologies) and 1% penicillin streptomycin (Gibco). The glands and tumors were digested overnight in a thermocycler maintained at 37 C with continuous rotation. For bulk RNAseq; the flash frozen whole mammary gland and tumors were homogenized and RNA was isolated using the RNeasy Mini Kit (#74104, Qiagen) according to manufacturer protocol. Single cell suspensions were input to a 10x Genomics Chromium machine to establish single-cell gel beads in emulsion (GEMs) for directed retrieval of approximately 5000-10000 cells scRNA-Seq libraries were prepared following the Single Cell 30 Reagent Kits v2 User Guide (Manual Part # CG00052 Rev A) using the following Single Cell 30 Reagent Kits v2: ChromiumTMSingle Cell 3' Library & Gel Bead Kit v2, PN-120237, Single Cell 3' Chip Kit v2 PN-120236, and i7 Multiplex Kit PN-120262 (10X Genomics). Bulk RNA-Seq were made using total RNA and the Illu-mina TruSeq mRNA sample preparation kit. scRNA-Seq Libraries were run on an Illumina NextSeq 500 paired-end reads. For bulk mRNA-Seq Paired end (2 50bp) sequencing was performed on the Illumina HiSeq 2000/2500 sequencer