GSM6250703: mRNA-derived cDNA Li98LN-G; Homo sapiens; OTHER
Human tumor lesions and non-involved adjacent tissues were digested as described in the material and method. Briefly, samples received mechanical and enzymatic digestion, filtration and the cell suspension was enriched in immune cells using a Percoll gradient, followed by a RBC lysis. Lymph nodes were digested mechanically and enzymatically followed by RBC lysis. Blood samples were processed by Ficoll followed by RBC lysis. Library was prepared using Chromium Single Cell 3ʹ v2 protocol/3ʹ  protocol (10x Genomics). Cellular mRNA and antibody-derived oligos were reverse-transcribed and indexed with a shared cellular barcode. Indexed cDNAs were then pooled and amplified by PCR according to the Chromium Single Cell associated protocol (10x Genomics) and with specific primers amplifying the antibody-derived tags. SPRI bead size selection was performed in order to separate both the mRNA-derived cDNA (>300bp) and the antibody-derived tagged cDNAs (180bp). For the mRNA derived cDNA library preparation, we further proceeded according to the manufacturer's instructions for Single Cell associated protocol (10x Genomics). For antibody-derived tagged library, we used the KAPA HiFI HotStart Library Amplification Kit (Roche) with the following primers and amplification program: -CITE-seq library: amplification was performed at 95°C for 3' followed by ten cycles at 95°C f¬¬or 20''; 60°C for 30''; 72°C for 20'' and final elongation 72°C for 5' with 5'-CAAGCAGAAGACGGCATACGAGATCGTGATGTGACTGGAGTTCCTTGGCACCCGAGAATTCCA-3' Small RNA RPI1 primer as i7 primer (Illumina). -HASH-seq library: amplification was performed at 95°C for 3' followed by ten cycles 95°C for 20''; 64°C for 30''; 72°C for 20'' and final elongation 72°C for 5' with 5'-CAAGCAGAAGACGGCATACGAGATCGAGTAATGTGACTGGAGTTCAGACGTGTGC-3' TruSeq D701_s primer as i7 primer (Illumina). The following 5'- AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTC-3' SI-PCR primer as i5 primer (Illumina) was used for both CITE- and HASH- seq library amplification. Following the final bead purification, all three libraries were pooled as 80% mRNA library, 10% ADT library and 10% HTO library before sequencing. OTHER: CITE-seq (Cellular Indexing of Transcriptomes and Epitopes by sequencing)