GSM8799854: mixed sample [1_S1]; Homo sapiens; RNA-Seq
PBMCs were separated using Ficoll density gradient centrifugation within 1 hour of blood collection and preserved in a freezing medium consisting of 90% fetal bovine serum (FBS) and 10% DMSO for future use. For non-responder patient were available biopsy samples of tumor tissue on days 3 and 21 after the first course of NAC. Each sample was collected using two 16-gauge needles. Samples were placed in the RPMI-1640 medium (Gibco, USA) and enzymatically digested no more than 20 minutes after collection. Tissues were washed extensively with 1× PBS with 200 U/mL penicillin. For collagenase/hyaluronidase treatment, tissue specimens were mechanically dissociated using a scalpel to remove vascular material, transferred to a solution of 300 U/ml collagenase and 100 U/ml hyaluronidase (STEMCELL Technologies, USA) in DMEM media, and then incubated at 37°C and 600×g for 30 min in a shaking incubator (BioSan TS-100, Poland). Once mucin clots formed, 5 U/ml of DNA hydrolyzing enzyme DNAase I (Sigma Aldrich, USA) was added to the suspension, followed by incubation under the same conditions for 10 min. To remove cell aggregates and debris, the cell suspension was filtered using a sterile cell strainer with a 70 μm pore diameter (Becton Dickinson, USA). Immediately after dissociation, the cell suspension was incubated with ROCK inhibitor (10 μM/ml) for 1 h, followed by cryopreservation in a solution of 95% FBS with 5% DMSO on ice. After isolation, cryovials with PBMCs or tumor cell suspensions were transferred in Mr.  Frosty Freezing Containers (Thermo Fisher Scientific, USA) containing isopropyl alcohol for gradient cooling and placed in a freezer, where they were stored for 24 hours at -80°C. Then the tubes were transferred to liquid nitrogen for long-term storage for up to 6 months at -196°C. For scRNA-seq, cells were removed from storage and rapidly thawed by immersion in a 37°C water bath with gentle agitation. Each cryovial was thawed individually until only a small amount of ice crystals remained. Samples were immediately transferred to round-bottom tissue culture tubes. Then, 10 ml of RPMI 1640 containing 10% FBS was added over 4 min with agitation, and the tubes were centrifuged immediately at 300×g for 5 min. The cells were then suspended in PBS containing 2% bovine serum albumin, followed by cell counting and viability assessment. The cellular viability of all PBMCs and tissue samples exceeded 75%, as evaluated by Calcein (BD Bioscience, USA) vs DRAQ7 (Thermo Fisher Scientific, USA) via flow cytometry. According to the manufacturer's protocol, 8 samples (6 PBMCs and 2 tumor cells suspensions) were labeled by Sample Tag and pooled for single-cell capture with the BD Rhapsody Single-Cell Analysis system (BD Bioscience, USA). 1,700 cells from each sample were included in the pool. Single-cell suspensions with PBS were loaded into microfluidic devices using the BD Rhapsody Single-Cell Analysis System (BD Bioscience, USA). Subsequently, the scRNA-seq libraries were constructed according to the protocol of the Immune Response Targeted Panel (Human) Single Cell RNA Library Kit (BD Bioscience, USA). Individual libraries were diluted and pooled according to the manufacturer's recommendations. Finally, Illumina NextSeq 2000 sequencing was performed using NextSeq 2000 P2 reagents (100 cycles).