title: GSM7006822: patient 1, scRNAseq; Homo sapiens; RNA-Seq
library_construction_protocol: The tumor tissues surgically resected from lung adenocarcinoma patients were minced with scissors and incubated with RPMI including 1 mg/ml BSA (Sigma), 1 mg/ml collagenase A (Roche) and 10 U/ml DNase 1 (Roche) f. The number of whole cells was counted using a hemocytometer, and the suspended cells were labeled with PE conjugated anti-human CD45 antibody (Invitrogen). Dead cells were removed by ReadiDrop PI staining (Bio-Rad). Tumor-infiltrating CD45+ immune cells were sorted by LE-SH800BC (Sony). Library was performed according to the manufacter's instructions (single cell 3' v3.1 protocol, 10x Genomics). Briefly, isolated 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 Silane DynaBeads, amplified by PCR and the apropiated sized fragments were selected with SPRIselect reagent for subsequent library construction. During the library construction Ilumina R2 primer sequence, paired-end constructs with P5 and P7 sequences and a sample index were added.