GSM5379892: Uterus, Restf/f PR+/Cre; Mus musculus; RNA-Seq
Whole uteri of Restf/f PR+/Cre cKO and control mice aged 5 months were isolated immediately after euthanasia using IACUCC approved methods, washed in cold 1X PBS. Uteri were processed by mincing the tissue and digesting in DMEM (Gibco, Waltham, MA), 10% FBS (Atlanta Biologicals, Flower Branch, GA), 1.5mg/ml collagenase (Gibco, Waltham, MA), and 2400U of DNaseI (NEB, Ipswich, MA) for 5 hours in a shaking water bath at 37°C. After digestion, cells were centrifuged at 300g for 5 minutes and treated with ACK lysis buffer followed by two washes with 1X PBS  and 2% FBS. Cells were passed through a 100um cell strainer and resuspended in 1X PBS and 2% FBS. Cells were passed through a 40um cell strainer and resuspended in 1X PBS and 2% FBS. Control samples (n=2) were pooled and cKO samples (n=2) were pooled. The KUMC genomics core determined concentration and viability using the Countess II FL Automated Cell Counter (Invitrogen, Waltham, MA) The RT reaction to generate 10X barcoded single stranded cDNA, in the single cell containing GEMs, is performed using an Eppendorf MasterCycler Pro thermal cycler. Post GEM-RT Cleanup is conducted by breaking the GEMS with 10X Recovery Agent to separate the aqueous phase from the Recovery Agent and Partitioning oil. A cleanup of the sscDNA containing aqueous phase is completed using Dynabeads MyOne Silane beads (Life Technologies 37002D). The second strand cDNA amplification is performed using the cDNA Amplification Mix on the Eppendorf MasterCycler Pro. The 3' gene expression library construction is initiated with fragmentation, end repair and A-tailing of the dscDNA followed by adapter ligation and a sample index PCR using the Illumina compatible indexed adapters in the Chromium i7 Multiplex kit (10X Genomics 120262). Validation of the single cell library is conducted using the Agilent Tapestation 4200 ScreenTape assay (Agilent 5067-5576). Single Cell RNA-seq (10X Genomics Chromium Single Cell 3' v3)