title: GSM5215218: HNSCC_Tum_ADT_2; Homo sapiens; RNA-Seq
library_construction_protocol: For HNSCC patient 1 (Pt1), there was single-cell suspensions from a juxtatumor sample and from a tumor sample. The 2 samples were stained with antibodies for cell sorting for 15 min at 4°C (Table 30). The samples were washed, and DAPI was added just before sorting. After sort, the different cell populations sorted were merged in defined proportions as detailed below for each of the 2 samples. The samples were stained with CITEseq antibodies (Table 30) at 4°C for 30min according to manufacturer's recommendations (Biolegend). Cells were washed, and the tumor sample was split in 2. In one of the samples, the primer specific for ADT was not added, to avoid the expansion by PCR of ADTs. This separation of the tumor sample in two was done to allow for comparison of tumor samples with or without ADT, because this was a new technology. We did not find any relevant difference between the samples with and without ADT, validating the absence of bias induced by this technological approach. Samples were then processed as recommended in 10X Genomics protocols.  For HNSCC patient 2 (Pt2), there was only one tumor sample available. Staining with antibodies for sort and with CITEseq antibodies were done simultaneously for 30 min at 4°C, before wash, DAPI staining and sorting. Sorting was performed using the purity and low-pressure mode, and a 100-μm nozzle on a BD FACS Aria III flow sorter for all samples.  The gating strategy is displayed in Fig S8. We excluded debris and doublets by FSC, SSC gating; dead cells by DAPI gating; red blood cells by CD235a staining (Pt2 only). All remaining cells were sorted in one of the 5 final population groups. Group 1 included CD45+CD3+ T cells; Group 2 included non-immune CD45- cells; Group 3 included MMAC and CD14+DC gated as CD45+CD3-CD19-CD56-CD11c+HLA-DR+CD14+; Group 4 included cDC1 and cDC2 gated as CD45+CD3-CD19-CD56-CD11c+HLA-DR+CD14- and was merged with pDC gated as CD45+CD3-CD19-CD56-CD11c-HLA-DR+CD123+; Group 5 included all remaining cells: CD45+CD3-CD19+ and/or CD56+, CD45+CD3-CD19-CD56-CD11c-HLA-DR+CD123-, and CD45+CD3-CD19-CD56-HLA-DR-.  The count provided by the flow sorter was used for subsequent processing. The population with the highest number of cells was also counted by trypan blue to ensure accuracy of the flow sorter counts. The final sample used for chip loading ScRNAseq contained 18000 (Pt1) or 16000 (Pt2) cells obtained by combining 3600 (Pt1) or 3200 (Pt2) cells from each of the 5 groups. The only exception was for the juxtatumor sample of Pt1, in which only 857 cells were available for the Group 4 of cells.   For the luminal breast cancer sample, the single-cell suspension from the tumor sample was stained with antibodies for cell sorting for 15 min at 4°C (Table 30). The sample was washed, and DAPI was added just before sorting. The gating strategy for DC included the following steps: (i) exclusion of debris and doublets by FSC, SSC gating; (ii) exclusion of dead cells by DAPI gating; (iii) selection of cells positive in CD45 BV570; (iv) exclusion of T cells, identified as CD3+ APC and CD19- Alexa700 and CD56- Alexa700; (v) exclusion of CD19+ Alexa700 and CD56+ Alexa700 cells; (vi) selection of CD11c- PECy5 cells, among which cells CD123+ BV650 and HLA-DR+ eFluor760 double positive were sorted as pDC; besides, selection of CD11c+ PECy5 and HLA-DR+ eFluor760 double positive cells, among which cells were classified according to CD1C PE and CD14 FITC: MMAC defined as CD1C- and CD14+ were excluded and all the other cells were sorted as cDC. Eventually, the DC sample used for ScRNA-seq included the pDC and the cDC. The CD45- cells, CD3+ cells, the MMAC and a mixed group of CD45+CD3- cells were also sorted independently but were not analyzed in the present study. Single-cell sequencing of final samples was performed using Single Cell 3' kit V1 (Breast cancer sample), v2 (HNSCC Pt1) and v3 (HNSCC Pt2) (10X Genomics), according to the manufacturer protocol (10X Genomics), including the CITE-seq libraries (HNSCC samples). Sample quality was checked with Bioanalyzer Agilent 2100 using a HighSensitivity DNA chip (Agilent Genomics). Samples were sequenced on an Illumina HiSeq (Breast cancer sample) and Novaseq (HNSCC samples) with a depth of sequencing of 50000 reads/cells (Breast cancer) and 100000 reads/cells (HNSCC samples).