title: GSM8793592: MC38 tum reaction 2, GEX; Mus musculus; RNA-Seq
library_name: GSM8793592
library_construction_protocol: Inguinal tumor-draining lymph nodes (TDLNs) and tumors were surgically removed with sterilized equipment. Lymph nodes were mechanically dissociated between two super-frosted microscope slides and washed into wells of 6-well plates containing cold PBS. Tumors were minced with scalpel blades and digested to single cell suspensions by incubation for 1h at 37C in tumor digestion media containing DMEM, 10%FBS, 2 mg/ml Collagenase IV (C5138, Sigma-Aldrich) and DNAse I (D5025, Sigma-Aldrich). Then, tumor lysates were filtered through a 100 μm filter into 50 ml conical tubes and filled with cold PBS. Tissue single cell lysates were centrifuged at 450 g for 5 minutes at 4C. Supernatants were discarded and pellets were resuspended in 5 ml ACK Lysing Buffer (118-156-101, Quality Biological), mixed well and kept on ice for 5 minutes. Lysis was stopped by filling the tubes with cold PBS. Samples were centrifuged again and finally resuspended in 1 ml cold PBS. Viable cells for downstream use were counted in a Vi-CELL cell counter (Beckman Coulter) at a 1:60 dilution. Library was performed according to the manufacter's instructions (single cell 10x 5' version 2 protocol, 10x Genomics). Briefly, cell suspensions were combined with master mix and loaded into the chip with gel beads to generate the gel bead-in-emulsion (GEM), which contains an Ilumina R1 primer sequence, Unique Molecular Identifier (UMI) and the 10x Barcode. Reverse transcription was performed on the poly-A RNA within each GEM. The pooled barcoded cDNA was then cleaned up with Silane DynaBeads and amplified by PCR. To prepare libraries from cDNA, SPRIselect beads were used to isolate the correct size fragments. Ilumina R2 primer sequence, paired-end constructs with P5 and P7 sequences, and a sample index were added during library construction.