title: GSM7670246: p53MUT; Mus musculus; RNA-Seq
library_name: GSM7670246
library_construction_protocol: Mammary glands from 8 week old virgin female mice were enzymatically digested and single cell suspensions were obtained following a standard protocol. Briefly, mammary glands were digested at 37°C for 1–2 hours in Epi-Cult-B medium (StemCell Technologies Inc) with 600 U/ml collagenase (Sigma Aldrich) and 200 U/ml hyaluronidase (Sigma Aldrich). After lysis of the red blood cells with NH4Cl, the remaining cells were washed with HBSS/0.02% w/v EDTA. Cells were then dissociated with 0.25% w/v trypsin, 0.2% w/v EDTA for 2 min by gentle pipetting, then incubated in 5 mg/ml Dispase II (Sigma Aldrich) plus 1 μg/ml DNase I (Sigma Aldrich) for 5 minutes, followed by filtration through a 40 μM cell strainer (BD Falcon). Library was performed according to the manufacter's instructions (Chromium Next GEM Single Cell 3' v3.1 protocol, 10x Genomics). Briefly, cells were resuspended in the master mix and loaded together with partitioning oil and gel beads into the chip to generate the gel bead-in-emulsion (GEM). The poly-A RNA from the cell lysate contained in every single GEM was retrotranscribed to cDNA, which contains an Ilumina R1 primer sequence, Unique Molecular Identifier (UMI) and the 10x Barcode. The pooled barcoded cDNA was then cleaned up with Silane DynaBeads, amplified by PCR and the appropiated sized fragments were selected with SPRIselect reagent for subsequent library construction. During the library construction Ilumina R2 primer sequence, paired-end constructs with P5 and P7 sequences and a sample index were added.