GSM5344033: NaïveLN scRNA; Mus musculus; RNA-Seq
Tumors were removed and cut into small pieces (~2 mm) with scissors. Tumor pieces were incubated in a digestion buffer containing 1 mg/ml collagenase type IV (Millipore Sigma, Darmstadt, Germany), in RPMI 1640 for 45 minutes (min) in a shaking incubator (Eppendorf New Brunswick Excella E25) at 37 °C and 200 RPM. Digested cell suspensions were passed through 100 μm mesh filters and were enriched for immune cells using Percoll (GE Healthcare Life Sciences, Pittsburgh, Pennsylvania, USA) density gradient centrifugation—— resuspended in 40% Percoll, layered on top of 60% Percoll and centrifuged for 30 min at 900x g at room temperature, without brake. Cells at the interface of the two Percoll layers were harvested and subsequently washed with FACS buffer in preparation for antibody staining. Cellular suspensions were loaded on a 10X Genomics GemCode Single-cell instrument that generates single-cell Gel Bead-In-EMlusion (GEMs) with Chromium Next GEM Single Cell 5' Reagent Kits v2（PN-1000263.The expression libraries were generated from the half of cDNAs with Library Construction Kit (PN-1000190) and the TCR libraries were constructed with the other half cDNAs by Chromium Single Cell Mouse TCR Amplification Kit(PN-1000254).Both of the libraries were sequenced using Illumina Novaseq6000 by Gene Denovo Biotechnology Co.(Guangzhou, China). Single-cell RNA-seq Single-cell TCR-seq
GEO
GSM5344033