GSM5211869: Patient61_BCR; Homo sapiens; RNA-Seq
Core biopsies were obtained under local anesthesia using a 12G core needle apparatus (Bard Magnum). Biopsies were placed immediately into a 50mL conical containing ice-cold DMEM and transferred on ice from the clinic to the laboratory (generally within 30 minutes of the procedure). A portion of the biopsy was immediately placed into formalin for subsequent paraffin embedding. The remainder of the biopsy was minced with a sterile scalpel into 2 mm pieces and placed in a gentleMACS C-tube (Miltenyi Biotech) containing 4.7mL of ice-cold DMEM. Enzymes from the Human Tumor Dissociation Kit (Miltenyi Biotech) were added to the sample according to the manufacturer's protocol for dissociation of tough tumors. After the enzymes were added, the C-tube was placed onto a gentleMACS Octo Dissociator (Miltenyi Biotech), the heating element was placed over the tube, and the 37C_h_TDK_3 program was initiated. Prior to completion of the dissociation protocol, two 70 μm cell strainers were each placed onto a 50mL conical on ice and pre-wetted with 3mL of heat-inactivated FBS. Upon completion of the dissociation protocol, the sample was poured through the first 70 μm cell strainer and the strainer was washed with 5mL of heat-inactivated FBS. The filtrate was then poured through the second 70 μm cell strainer and the strainer was washed with 5mL of heat inactivated FBS. The sample was centrifuged at 300xg for 7 minutes at 4C. The supernatant was removed, and the sample was resuspended in 2mL of DMEM and transferred to a microcentrifuge tube on ice. An aliquot of the sample was taken for cell counting via a hemocytometer and the remaining sample was centrifuged at 300xg for 10 minutes at 4C. The supernatant was removed, and the sample was resuspended in 80μL FACS buffer per 10^7 cells. Miltenyi anti-human CD45 microbeads were added to the sample (20μL per 10^7 cells), and the sample was incubated at 4C covered with foil for 15 minutes. After incubation, 1mL FACS buffer per 10^7 cells was added to the sample and pipet mixed. The sample was centrifuged at 300xg for 10 minutes at 4C. The supernatant was removed, and the sample was resuspended with 500μL FACS buffer and transferred to a 15mL conical. The sample was placed in a pre-cooled AutoMACS tube holder and the Miltenyi AutoMACS Possel_S program was initiated. At this time, 10X Genomics reagents required for running the Single Cell 5' v1 Assay were removed from their storage conditions to equilibrate at room temperature for 30 minutes. Upon completion of the AutoMACS program, the CD45-positive fraction was centrifuged at 300xg for 7 minutes at 4C. The sample was resuspended in 50μL of DMEM and a cell count was performed via a hemocytometer. If possible, the sample was diluted to 1000 cells/μL and the sample was processed according to the 10X Genomics protocol for the Single Cell 5' v1 assay with a targeted 10,000 cell recovery (or maximum recovery possible with the number of cells in the sample). Subsequent gene expression libraries and T-cell receptor (TCR)/B-cell receptor (BCR) enriched libraries were prepared according to the 10X Genomics Single Cell 5' v1 protocol. Libraries were sequenced using paired-end 150bp reads to an average 60-70% sequencing saturation (as calculated by 10X Genomics Cellranger v4.0.0) on an Illumina Novaseq.