title: GSM6862039: SOCS3WT_KO_Tcells_scRNAseq; Mus musculus; RNA-Seq
library_construction_protocol: MC38 tumor mass were excised and minced and digested with 0.25% Liberase TM (Merck) and 20 kU/mL DNase I (Sigma-Aldrich, St Louis, MO) at 37 °C for 30 minutes with gentle shaking. Cells were passed through a 100-μm filter and subjected to 40% Percoll centrifugation to remove debris and epithelial cells from the surface of the solution. Red blood cell lysis was performed if necessary, and the cells were washed twice with phosphate-buffered saline (PBS). The cells were incubated with anti-CD3e+-Brilliant violet-421 , anti-TCRb+-APC antibodies, and Fixable viabily dye -eFlour 780 in the presence of TotalSeq -C0301, C0302, C0303 or C0304 anti-mouse Hashtag antibodies in each mouse sample. CD3e+ TCRb+ cells were isolated by FACS AriaIII. Library was performed according to the manufacter's instructions (Chromium Next GEM SIngle Cell 5'Reagent Kits v2, 10x Genomics). Briefly, T 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 reversely transcribed to cDNA with Gel Bead primers containingan Illumina TruSeq Read1 sequence, a  10x Barcode, a unique Molecular Identifier (UMI), and template switch oligo (TSO), and poly(dT) primers.  Simultaneously in the same partition, the Gel Bead primer captures the cell surface protein Feature Barcode conjugated to HashTag antibodies containing a Nextera Read 2 , a Feature Barcode, and capture sequence, and produces 10x Barcoded DNA with Feature Barcode of Hash Tag antibodies. The pooled barcoded cDNA was then cleaned up with Silane DynaBeads, amplified by PCR and the apropiated sized fragments were selected with SPRIselect reagent for subsequent library construction. During the library construction Ilumina R1 primer sequence, paired-end constructs with P5 and P7 sequences and a sample index were added.