GSM8774330: 4T1 tumors control sgRNA; Mus musculus; RNA-Seq
Tumors cut into small pieces were digested in digestion buffer (HBSS with Mg2+/Ca2+ supplemented with 2 mg/ml collagenase type I and 40 U/ml Dnase I) and incubated at 37C for 45 min. cells were washed and resuspended in PBS and stained with Viability Dye, washed and resuspended in FACSbuffer (HBSSwo, 2% v/v FCS, 5 mM EDTA). Live cells were sorted using FACs Jazz (Becton Dickinson) according to the presence or absence of the mCherry signal. 4T1 cells were used as negative control. Sorted cells were  washed in HBSS-?- and resuspended in HBSS-/- supplemented with 0.04% w/v BSA. Library construction was performed according to the manufacturer's instructions )single cell 3' v2 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 retrotranscripted 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 Silan DynaBeads, amplified by PCR and the appropriately sized fragments were selected with SPRIselect reagent for subsequent library construction. During the library construction of Ilumina R2 primer sequence, paired-end constructs with P5 and P7 sequences and a sample index were added