title: GSM8163729: Muscle, Icos-/- NOD, CD45-, PROX [4_6|008|CD45-]; Mus musculus; OTHER
library_construction_protocol: Tissues were frozen in chilled in liquid nitrogen for ~ 1,5 min. Six-micrometer-thick cryosections were prepared using the GeoMx Digital Spatial Profiler (DSP) Whole Transcriptome Atlas (WTA) slide prep protocol from NanoString Technologies. Sections were incubated with(WTA RNA-ISH probes conjugated with UV-photocleavable linkers following standard ISH protocols. Entire slides containing Icos+/+ NOD and Icos-/- NOD sections were imaged at ×20 magnification, and Regions of Interest (ROIs) (4 Icos+/+ NOD and 14 Icos-/- NOD) were selected. The GeoMx DSP segmentation tool allowed the separation of different areas of illumination (AOI) on the basis of morphology markers (CD45 and desmin). This strategy allowed the analysis of RNA expression from myofibers (CD45-desmin+) from Icos+/+ NOD (n=4) and from Icos-/- NOD mice, the latter being non-adjacent (designated as “Icos-/- NOD mice PROX”) (n=6) or adjacent (designated as “Icos-/- NOD mice ADJ”) (n=8) to immune cell infiltrate clusters (CD45+desmin-). Regions of interest within the tissue were exposed to UV light and oligo barcodes were physically aspirated from the tissue and collected into microtiter plates by the GeoMx® Digital Spatial Profiler (DSP) platform. DSP protocol detailed information can be found in Merritt et al. Nature Biotech 2020 (doi: 10.1038/s41598-020-63539-x). Each collection of oligo tags from one well (corresponding to an AOI from the tissue section) was indexed with i7xi5 unique dual indexes using GeoMx SeqCode primers with 18 cycles of PCR.  After PCR, indexed AOIs were pooled and purified by AMPure XP PCR purification. Pooled libraries were sequenced at 2 × 27 base pairs and with the dual-indexing workflow on an Illumina NovaSeq 6000.