title: GeoMx_dlbcl
organism: Homo sapiens
description: Protocols: One to three lung, breast and/or DLBCL tissue sections of 5 micrometer thickness were placed on each slide, sectioned from FFPE blocks. Slides were processed according to the manufacturer's instructions. Tissue sections were cut at 5 um thickness. They were baked at 60°C for 2 hours. Sections were stained on a BOND-RXm fully automated multiplexing immunohistochemical stainer (Leica Biosystems; Wetzlar, Germany) using the following immunofluorescent antibodies: for Breast and Lung, PanCK - AF532 (Clone AE1 + AE3 from Novus) diluted at 1/50, CD3 - AF647  (Clone CD3-12 from Biorad) diluted at  1/100, CD68 - AF594  (Clone KP1, from Santa Cruz) in concentration 0.25 ug/ml and SYTO 13  nuclear stain diluted at 1:10000 (Nanostring) in Buffer W for 1 h at room temperature; for DLBCL, CD20 - AF594 (Clone IGL/773 from BioTechne) diluted at 1/100, CD3 - AF647 (Clone CD3-12 from Biorad) diluted at 1/100, CD68 - AF488 (Clone KP1 from e-BioMed) at concentration 0.25 ug/ml and SYTO 83 nuclear stain diluted at 1:100000 (Invitrogen) in Buffer W for 1 h at room temperature.  Regions of interest (ROIs) were selected by pathologists, and segmented into areas of illumination (AOI-s) based on immunofluorescent markers (Supplementary Tables 3-4). The first segment was defined as double positive for CD3 and CD68 with the purpose of excluding autofluorescent structures (such as elastin fibers in lung) or debris, and it was not collected. For Lung and DLBCL samples, the CD3-AOI-s were collected first, followed by CD68-AOI-s. For Breast samples, CD68-AOI-s were collected first, followed by CD3-AOI-s. PanCK-AOI-s and CD20-AOI-s were collected third for solid cancers and DLBCL respectively. For five and four ROI-s in breast and lung replica samples respectively, only two segments of PanCK+ and PanCK- were collected in that order, ignoring other markers. The remaining cells showing SYTO nuclear expression in the absence of any cytoplasmic marker were collected as the fourth marker-negative (Other) segment in BC. All the remaining surface of ROIs which had not been collected into any AOI was collected as marker-negative (Other) segments for Lung and DLBCL, independently of the presence of nuclei. With the exception of the Other segment of Lung and DLBCL, a segment was collected when it contained at least 50 cells. About 24 such AOI-s per patient were collected with AOI size varying from about 500µm2 to 300,000µm2 with an average size of 60,000µm2. Library preparation and sequencing was performed according to the manufacturer's instructions and kits.  All Breast, Lung and DLBCL samples were assayed on the Nanostring GeoMx Digital Spatial Profiler (DSP) platform using the Whole Transcriptome Atlas (WTA) probe panel with NGS readout.
attributes.tag: isolate
attributes.value: not applicable
attributes.tag: disease
attributes.value: cancer
attributes.tag: organism
attributes.value: Homo sapiens
attributes.tag: organism
attributes.value: Homo sapiens
attributes.tag: collection date
attributes.value: not collected
attributes.tag: scientific_name
attributes.value: Homo sapiens
attributes.tag: common name
attributes.value: human
attributes.tag: organism part
attributes.value: various
attributes.tag: developmental stage
attributes.value: adult
attributes.tag: geographic location (country and/or sea)
attributes.value: not collected