GSM5452695: Barcoded, pooled single B cells [J_13]; Homo sapiens; RNA-Seq
Tumours were cut into ca. 1cm3, enzymatically digested in RPMI medium (Gibco, Paisley, UK) with 1 mg/µL collagenase type IV (Gibco Life Technologies, Grand Island, USA) and 12,6 µg/mL recombinant human DNase (Pulmozyme, Roche, Woerden, the Netherlands) for 30 minutes at 37 °C or overnight at room temperature. Digests were filtered using 70 µm cell strainers (Falcon) and enriched for peripheral blood mononuclear cells (PBMCs) using Ficoll-Paque PLUS (GE Healthcare Life Sciences, Marlborough, MA, USA). Cells were stored in liquid nitrogen until cell sorting. For cell sorting, tumour digests were thawed and washed with PBS and incubated with Zombie Aqua (1:100, Biolegend, San Diego, USA) for 15 minutes at room temperature. Samples were washed and stained with anti-CD27 APC-efluor 780 (clone O323; 47-0279-42; eBioscience) and either CD19-PE (clone HIB19; 12-0199-41; eBioscience), CD19-BV421 (clone HIB19; 562440; BD Biosciences), CD19-APC (clone HIB19; 17-0199-41; eBioscience) or CD19-PE-cy7 (clone HIB19; 25-0199-41; eBioscience) for 45 minutes at 4°C. Cells were washed and filtered using a 35 µm strainer (Falcon). Patient samples were pooled to minimize differences due to plate effects and sorted on a Beckman Coulter MoFlo Astrios. Each well contained 2 µl lysis buffer (0,2% Triton X-100 (Sigma-Aldrich) and 2U RNase inhibitor (Takara)) with 1 µl 10 µM oligo-dT primer and 1 µl 10 mM dNTP mix (Thermo Scientific). After sorting, the plate was spun down and incubated at 72°C for 3 minutes. SmartScribe reverse transcriptase (Westburg-Clontech) and a template switching oligo (BC-TSO) were used to generate cDNA. Next, an exonuclease step was performed using 1:400 dilution of Exonuclease I.  A PCR preamplification step was done with KAPA HiFi HotStart Ready Mix (Roche Diagnostics, 23 cycles in experiment 1 and 25 cycles in experiment 2) and a custom-made PCR primer. The cDNA samples were purified using Ampure XP beads (Beckman Coulter) in a ratio of 0,8:1 (Ampure bead : cDNA). Samples were analysed on a 2100 Bioanalyzer using a PerkinElmer LabChip GX high-sensitivity DNA chip (Agilent) and on a Qubit™ 4 Fluorometer (ThermoFisher Scientific) according to manufacturer's instructions. 500 pg of each sample was tagmented and N7xx and S5xx index adapters were used for barcoding according to the Illumina Nextera XT DNA sample preparation kit (Illumina). Thereafter, samples were purified with Ampure XP beads (ratio 0,6 Ampure : 1 cDNA) and analysed on a 2100 Bioanalyzer. Samples were equimolar pooled (4 nM) and samples were sequenced on an Illumina Nextseq500 2500 using 75 bp paired end reads. Modified SMARTseq2