title: Single nuclei RNA sequencing of patient sample: Sample UMEL_3_2
design: Single-nuclei suspensions were generated using methods published in Slyper et al. (Nature Medicine, 2020) with modifcations. Frozen tissue specimens with 2-10 mm edge length were embedded in optimal cutting temperature (OCT) compound (Tissue-Tek, Sankura; #4583) on dry ice. Samples were then mounted on sample holders and excess OCT was trimmed away using a blade leaving ~5 mm OCT around each side of the tissue. Fine needle aspiration biopsies were directly mounted on sample holders of the cryostat using a small amount of OCT. Multiple 20 µm tissue curls were cut per tissue and collected in pre-cooled 5 ml tubes (Eppendorf, Hamburg, Germany) ensuring no thawing while transferring and stored on dry ice until processing. Salt-tris buffer with Tween-20 (TST) was used for dissociation followed by a high-volume wash in salt-tris buffer (ST). 0.9-1.5x103 nuclei were then loaded in ST buffer with or without RNAse inhibitor using a Chromium controller and chromium reagents (10X Genomics) for 3' (#1000075) or 5' capture (#1000006 and #1000263) as indicated.