Human tumor dissociation kit

Fresh tissue was collected into RPMI 1640 medium supplemented with 2% human serum (Sigma), cut into 1 mm² pieces, and enzymatically digested for 20min at 37°C using the Human Tumor Dissociation Kit (Miltenyi Biotec) in the presence of 10μM ROCK inhibitor Y-2763 (Sigma). Cell suspension was passed through 70μm cell strainers and centrifuged for 7min at 450g at 4°C. Supernatant was removed and cells were subject to ACK Lysing Buffer (Life Technologies) for 2min on ice, centrifuged for 7min at 450 g at 4°C, and resuspended in RPMI 1640 supplemented with 2% human serum (Sigma). The single cell suspension was stained with Zombie Violet in PBS (Invitrogen) for 10min on ice and subsequently with antibodies against human CD326, CD45, and CD235a (Biolegend) in RPMI 1640 medium supplemented with 1% human serum in the presence of 10μM Y-2763 for 15 min on ice. Zombie Violet⁻ CD235a⁻ CD45⁻ CD326⁺ cells were bulk sorted into 1.5ml Eppendorf tube containing 1× TD buffer, 2.5μl Tn5 (Illumina), 0.1 % NP40, 0.3× PBS in a 50μl reaction volume for ATAC-seq as described above. Using the identical gating scheme, single cells were sorted into Eppendorf twin-tec PCR plates containing 10μl TCL lysis buffer (Qiagen) supplemented with 1% beta-Mercaptoethanol and processed for scRNAseq as described above.

A single cell suspension of surgically resected human pituitary tumors was obtained by mechanical and enzymic digestion using a gentleMACS dissociator, and human tumor dissociation kit (Miltenyi Biotec Inc., Germany, Cat# 130-095-929). Library generation was performed on the 10x Genomics Chromium Controller following the manufacturer’s protocol for the v3 reagent kit (10x Genomics). In brief, cell suspensions were loaded onto a Chromium Single Cell A Chip, aiming for 10,000 cells per channel for generation of single-cell gel bead-in-emulsions (GEMs), following which reverse transcription was performed. The resulting post-GEM reverse transcription product was then cleaned using DynaBeads MyOne silane beads (Thermo Fisher Scientific, Waltham, MA). The cDNA was amplified, cleaned and quantified, then enzymatically fragmented and size selected prior to library construction. Libraries were quantified by KAPA quantitative PCR for Illumina adapters (Roche, Pleasanton, CA) and size was determined by Agilent TapeStation D1000 tapes. Libraries were sequenced on a NextSeq 500 sequencer (Illumina, San Diego, CA).

Tumors were collected post-operatively from patients who signed consent to use their biospecimen for research under IRB s16-00122. Each sample was washed in PBS and cut into 4–5-mm³ pieces, of which 2–3 were reserved for spatial transcriptomics (see below). The remainder was dissociated for scRNA-Seq using the Miltenyi human tumor dissociation kit according to manufacturer’s instructions. Red blood cell lysis was performed in ACK lysis buffer for 3 minutes. Cells were counted and viability was assessed by trypan blue on a hemocytometer. For samples with low viability (<50%), dead cells were removed using the Miltenyi dead cell separator. Single-cell encapsulation and library preparations were performed using the inDrop platform according to manufacturer’s instructions^{76 (link)}. Libraries were sequenced on an Illumina NextSeq and reads aligned using the inDrop pipeline previously described^{77 (link)}. To exclude cells with low quality transcriptomes from analysis, cells with fewer than 500 UMIs or more than 30% mitochondrial or ribosomal reads were filtered out. The Seurat v.4.0.3 single-cell transformation^{78 (link),79 (link)} was used to normalize and center the data, and to identify variable genes.