GSM6755347: FFPE1 | 002 | CD30; Homo sapiens; OTHER
For GeoMx DSP sample collections, entire slides were imaged at 20X magnification. ROIs were segmented using CD30 and CD4 markers. CD30+ large cells were defined as HRS cells (CD30+). CD4+ cells within 20μm from CD30+ HRS cell were defined as “contour”. The remaining CD4+ cells were defined as “remainder”. A total of 12 AOIs from 4 ROIs were segmented and exposed to 385 nm light (UV), releasing the indexing oligonucleotides which were collected with a microcapillary and deposited into a 96-well plate for subsequent processing. The indexing oligonucleotides were dried overnight and resuspended in 10 μl of DEPC-treated water. Sequencing libraries were generated by PCR from the photo-released indexing oligos and AOI-specific Illumina adapter sequences. Unique i5 and i7 sample indexes were added. Each PCR reaction used 4 μl of indexing oligonucleotides, 4 μl of indexing PCR primers, 2 μl of Nanostring 5X PCR Master Mix. Thermocycling conditions were 37 °C for 30 min, 50 °C for 10 min, 95 °C for 3 min; 18 cycles of 95 °C for 15 s, 65 °C for 1 min, 68 °C for 30 s; and 68 °C for 5 min. PCR reactions were pooled and purified twice using AMPure XP beads (Beckman Coulter, A63881), according to the manufacturer's protocol.