GSM9012006: TRISS036, CD3 T cells, sTCRseq; Homo sapiens; OTHER
Tumor tissue from endometrial cancer patients was collected during primary surgery. Tumors were minced into pieces and enzymatically digested with RPMI (Gibco) containing 1 mg/ml Collagenase type IV (Sigma) and 13 U/ml Pulmozyme (Roche), using the GentleMACS Dissociator (Miltenyi), program h_tumor_01.01. Digestion was either done overnight at room temperature or for 30 minutes at 37 °C. Digests were filtered using 70 µm cell strainers and cells were enriched using Ficoll-Paque PLUS (GE Healthcare). After washing with PBS, digests were cryopreserved in fetal bovine/calf serum supplemented with 10% dimethylsulfoxide (DMSO), and stored at liquid nitrogen until use. The day of the experiment, tumor digests were thawed in 2 mL of FCS, centrifuged at 300 g for 5 min, supernatant was discarded, and pellet was resuspended in 0.5 mL of RPMI + 10% FCS. After 1.5h incubation at 37°C/5% CO2, samples were stained and incubated with a CXCR5 PerCP-Cy5.5 antibody for an additional 0.5h at 37°C. After a total of 2h recovery at 37°C/5% CO2, samples were centrifuged and resuspended in 100 µl PBS/2% FCS containing the appropriate surface antibodies. Samples were incubated for 30-45 minutes on ice (in dark) with the following antibodies: CD45-BV605, CD8-APC Cy7, CD4-PE, CD19-BV421, PD1-APC, CD3-PE-Cy7 (Table S1). 3 µl of each antibody was used per 1x10e6 cells. Samples were washed twice with PBS/2% FCS and filtered using a 35 µm strainer (Falcon) before sorting on a Beckman Coulter MoFlo Astrios cytometer. UltraComp eBeads (Thermo Fisher Scientific) were used as compensation controls. Before sorting, Propidium Iodide was added to exclude dead cells. Sorting was done in 1.5 mL Low Bind DNA tubes (Eppendorf) in 150 µl PBS 0.04 % BSA. CD45+ CD3- CD19+ (B cells), CD45+ CD3+ CD19- CD8+ (CD8 cells) and CD4+ (CD4 cells) or CD45+ CD3- CD19- cells were sorted. Sorted cells were centrifuged and resuspended at a concentration of 727 – 2121 cells/µl (total 16.5 µl), in PBS 0.04% BSA, depending on the total number of sorted cells and sample. When the target number of cells from a given population could not be reached for any sample, those cells were jointly loaded onto the chromium chip with cells from the CD3- CD19- CD45 population. As such, the fraction of single cells sequenced does not quantitatively reflect the immune cell composition per sample (Fig S1). Subsequent steps were performed according to manufacturer's protocol (10x genomics): 'chromium next GEM single cell 5' reagents kit v2 (dual index)'. For the cDNA amplification step, 13 cycles were performed according to the manufacturer's instructions. For each sample, both the V(D)J library as well as the gene expression (GEX) library was constructed. All cDNA quality control and quantification steps were done with the Qubit 4 Fluorometer (Thermo Fisher) and the Agilent 4200 Tapestation (with D5000 and high sensitivity D5000 screen tapes). For the sequencing, V(D)J and GEX libraries were pooled at a 1:4 molar ratio. 1.5 pM of the superpool was sequenced on a NextSeq 500 (Illumina inc., San Diego, 521 CA, USA). A 150 bp NextSeq high-output reagent kit was used for the sequencing (paired end). Library construction was performed according to manufacturer's protocol (10x genomics): 'chromium next GEM single cell 5' reagents kit v2 (dual index)'. For the cDNA amplification step, 13 cycles were performed according to the manufacturer's instructions. For each sample, both the V(D)J library as well as the gene expression (GEX) library was constructed. All cDNA quality control and quantification steps were done with the Qubit 4 Fluorometer (Thermo Fisher) and the Agilent 4200 Tapestation (with D5000 and high sensitivity D5000 screen tapes). For the sequencing, V(D)J and GEX libraries were pooled at a 1:4 molar ratio. 1.5 pM of the superpool was sequenced on a NextSeq 500 (Illumina inc., San Diego, 521 CA, USA). A 150 bp NextSeq high-output reagent kit was used for the sequencing (paired end).