GSM7422176: 16_S100B_102; Homo sapiens; RNA-Seq
day1:TMA slides were processed using the Nanostring GeoMx DSP manual slide preparation GeoMx®-DSP protocol (MAN-10150-01).Antigen retrieval of the FFPE tissue was performed for 20 minutes followed by deparaffinization and rehydration.Then, the slides were incubated with proteinase K for 20 minutes and RNA probes were placed on slides for overnight in situ hybridization. day2:slides were washed to remove off-target probes for 90 minutes. Morphology markers: CD68 for macrophage compartment (Cy5/666 nm, excitation 645/19 nm, emission 683/30 nm), CD45 for lymphocyte compartment (Texas Red/615 nm, excitation 588/19 nm, emission 623/30 nm), S100B (Cy3/568 nm, excitation 538/19 nm, emission 564/15nm) for tumor compartment to identify the compartments and SYTO 13 for nuclear stain, were added for 1-hour incubation. Slides were loaded onto the GeoMx DSP instrument and after scanning, ROI (region of interest: i.e., CD45, CD68 and S100B) selection and probe collection were performed according to the GeoMx®-DSP user manual protocol (MAN-10088-03). Samples were collected in a 96-well probe aspirate collection plate and each ROI representing a compartment from a patient tumor core was collected in a well. GeoMx®-DSP user manual (MAN-10117-01); Each GeoMx DSP aspirate in the plate contains photocleaved DNA oligos comprised of an RNA analyte identifier, a unique molecular identifier (UMI) barcode, and primer binding sites. Prior to purification, PCR reactions were pooled into three mixtures, separating CD68, CD45 and S100B ROIs to generate three pooled libraries. The libraries were pooled in a biased manner designed to target the smallest ROI pool with 6 parts of reads for CD68, 3 parts of reads for CD45 and 1 part of reads for S100B. Illumina i5 x i7 system adapter sequences and unique dual sample indices are added when PCR is performed on the probe aspirates. Each photocleaved oligo in the GeoMx DSP collection plate contains a readout tag sequence identifier (RTS ID) that identifies the target. It also includes a unique molecular identifier (UMI), to remove PCR duplicates when converting reads to digital counts. Read 1 (SPR1) and Read 2 (SPR2) are binding sites for Illumina sequencing primers. The GeoMx Seq code primers that hybridize to SPR1 and SPR2 contain i5 or i7 indexing sequences as well as P5 or P7 sequences for binding to Illumina flow cells. Sequencing was performed in both lanes of an Illumina Nova-Seq 6000 S2 flow cell resulting of 3 billion reads