GSM5823472: BC11_TIL; Homo sapiens; RNA-Seq
Blood and tumors were obtained from patients undergoing surgical resection as treatment for breast cancer. Blood specimens were collected by venipuncture into EDTA collection tubes.  Peripheral blood mononuclear cells were isolated from whole blood by Ficoll-Hyapque density gradient centrifugation. Surgical specimens were collected in RPMI 1640 medium supplemented with 10% fetal bovine serum and 1% penicillin and streptomycin. Single cell suspensions were generated from tissues by manually micing tissues and incubating the resulting tissue fragments in serum-free RPMI 1640 with 50 ug/ml of Liberase DL and 1 mg/ml DNase I for 30 minutes. Live CD45+ cells (i.e. all immune cells) were then sorted using a MoFlo Astrios High Speed Sorter (Beckman Coulter). Cell hashing using oligonucleotide conjugated antibodies was performed to multiplex peripheral blood and tumor specimens from the same patients as indiciated in the sample records. Samples were run using either 3'- or 5'-based single-cell RNAseq as described in the samples records. Libraries were prepared as per the manufacturer's protocol for 3'- and 5'-based single-cell RNAseq. For cell hashed samples, CITEseq libraries were generated as descibed by Stoeckius et al (Nature Biotechnology, 2017).