Homo sapiens
Protocols: Fresh CD4 positive T cells extracted from tumor and healthy tissue were harvested, washed with 1 PBS and resuspended in PBS and 0.04% bovine serum albumin. Cell were then counted using an automated counter (Countess II Thermo Fisher) and cell viability was assessed by Trypan blue exclusion. Samples were selected according to cell viability only samples with a viability above 85% were used for single-cell analyisis. After quantification cells were resuspended at 1e106 cells per ml in PBS and 0.04% bovine serum albumin. Cellular suspensions (5000 cells per sample) were loaded on a Chromium 10X Instrument (10x Genomics, Pleasanton, CA, USA) to generate single-cell GEMs. Cellular suspensions (5000 cells per sample) were loaded on a Chromium 10X Instrument (10x Genomics, Pleasanton, CA, USA) to generate single-cell GEMs. Single-cell RNA-Seq libraries were prepared using Chromium™ Single Cell 3' Library & Gel Bead Kit v2 (16 rxns PN-120237; 10x Genomics). GEM-RT was performed in a Veriti© 96-Well Thermal Cycler (Thermo Fisher; PN-4375786): 55 C for 2 h, 85 C for 5 min; held at 4 C. After RT, GEMs were broken and the single-strand cDNA was cleaned up with DynaBeads MyOne Silane Beads (Thermo Fisher Scientific; P/N 37002D) and SPRIselect Reagent Kit (0.6 SPRI; Beckman Coulter; P/N B23318). cDNA was amplified using the Veriti© 96-Well Thermal Cycler: 98 C for 3 min; cycled 14 : 98 C for 15 s, 67 C for 20 s, and 72 C for 1 min; 72 C for 1 min; held at 4 C. Amplified cDNA product was cleaned up with the SPRIselect Reagent Kit (0.6 SPRI). Indexed sequencing libraries were constructed using the reagents in the Chromium™ Single Cell 3' Library Kit, following these steps: (1) fragmentation, end repair and A-tailing; (2) adapter ligation; (3) post-ligation cleanup with SPRIselect; (4) sample index PCR and cleanup. The barcode sequencing libraries were quantified by quantitative PCR (KAPA Biosystems Library Quantification Kit for Illumina platforms P/N KK4824).
isolate
not applicable
organism
Homo sapiens
individual
CRC_2
age
57
sex
male
organism part
colon
sampling site
normal tissue adjacent to neoplasm
cell_type
T cell
phenotype
CD4 positive
disease
colorectal cancer
ENA-FIRST-PUBLIC
2020-03-04
ENA-LAST-UPDATE
2018-07-16