GSM5138682: Patient2P3; Homo sapiens; RNA-Seq
Following pathological examination, tumor material was kept on ice in DMEM/F12 + GlutaMAX (GIBCO, Invitrogen Life Technologies), 1% HEPES, 1% penicillin-streptomycin from that point onwards. Tumors were mechanically dissociated using sterile scalpels and enzymatically digested with 25 μg/ml DNase I (Roche) and 15 Wünsch units TH Liberase /ml (Roche) diluted in PBS at 37 ⁰C for 35 minutes, followed by mashing through a 70 µm filter (BD Falcon) while adding DMEM/F12 + GlutaMAX (GIBCO, Invitrogen Life Technologies), 1% Hepes, 1% Penicillin-Streptomycin. Tumor cells were blocked in 80% FACS buffer (5 mM EDTA in PBS supplemented with 5% fetal calf serum / 20% serum mix (50/50 normal goat serum (monx10961, Monosan) and FcγII/III receptor blocking serum 2.4G2 (kind gift from L. Boon, Bioceros, The Netherlands)) for 10 minutes on ice before labeling with EpCAM PE (1B7, eBioscience), CD14-FITC (MɸP-9, bdBiosciences) labeled antibodies for 40 minutes on ice. DAPI was added as a cell death marker, and cells were sorted either on a FACS AriaII Special Ordered Research Product (BD Biosciences), on a FACS Jazz (BD biosciences), or on a FACS Fusion (BD biosciences). A broad FSC SSC gate was followed by a gate excluding doublets, after which the DAPI negative cells were selected. These DAPI negative cells were sorted into 384-well plates and further processed for single cell sequencing. To obtain enough cancer cells and macrophages, additionally EpCAM positive and CD14 positive cells were selected and index-sorted into 384-well plates and further processed for single cell sequencing. For 3 out of 10 patients, only EpCAM positive and CD14 positive cells were isolated and sequenced. Single-cell mRNA sequencing was performed using Sort-seq as described in Muraro et al. (Cell Systems 2016). Briefly, cells were sorted into 384 well plates containing a lysis buffer and barcoded RT primer containing a T7 promoter. Reverse transcripion and second strand synthesis were performed individually for each single cell. Afterwards cells were pooled and IVT was performed. Libraries were prepped according to the Illumina small RNA protocol. Single-cell libraries were sequenced on an Illumina NextSeq500 with 75bp paired end reads.