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. 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). 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).
ENA first public
2021-10-20
ENA last update
2021-10-20
External Id
SAMEA7298092
INSDC center alias
Istituto Nazionale Genetica Molecolare Romeo ed Enrica Invernizzi, 20122 Milan, Italy. Department of Medical Biotechnology and Translational Medicine, Universita degli Studi di Milano, 20133 Milan, Italy.
INSDC center name
Istituto Nazionale Genetica Molecolare Romeo ed Enrica Invernizzi, 20122 Milan, Italy. Department of Medical Biotechnology and Translational Medicine, Universita degli Studi di Milano, 20133 Milan, Italy.
INSDC first public
2021-10-20T00:15:10Z
INSDC last update
2021-10-20T00:15:10Z
INSDC status
public
Submitter Id
E-MTAB-7006_2:scSQ_2093
age
54
broker name
ArrayExpress
cell type
T cell
common name
human
disease
colorectal cancer
individual
CRC_3
organism part
colon
phenotype
CD4 positive
sample name
E-MTAB-7006_2:scSQ_2093
sampling site
normal tissue adjacent to neoplasm
sex
male