GSM9230317: PyMT_PCBP1_KO(2); Mus musculus; RNA-Seq
Sized-matched mammary tumors from PyMT PCBP1+/+ and PyMT PCBP1–/– females were collected and flash frozen in liquid nitrogen. Tissues were chopped and cell membrane were lysed in NP40 lysis buffer (10mM Tris-HCl pH7.4, 10mM NaCl, 3mM MgCl2, 1mM DTT, 0.1% NP40, 1U/µL RNAse inhibitor) using a mini bead mill and crude nuclei suspensions were filtered through 70 µm strainers. Nuclei were spun, resuspended in pre-chilled 1% BSA PBS, stained with 10µg/mL 7AAD, and positive nuclei were collected by FACS in 1% BSA PBS. Single-nucleus suspensions were prepared from mammary tumors, evaluated for quality (Cellometer, Nexelom Bioscience), and adjusted to 1,000 nuclei/µL. Libraries were generated using the Chromium Next GEM Single Cell 3′ Library & Gel Bead Kit v3.1 (10x Genomics, Cat. no. CG000315) according to the manufacturer's protocol. Nuclei were loaded onto a Chromium Next GEM Chip G and partitioned on a Chromium Controller (10x Genomics). Barcoded cDNA was amplified and converted into gene expression libraries following the Chromium Next GEM Single Cell 3′ protocol. Libraries were sequenced on an Illumina NovaSeq 6000 S4 flow cell (2 × 150 bp, ~300 million paired-end reads per library) at the VANTAGE facility (Vanderbilt University Medical Center).