GSM8229212: DSP-1012340055901-B-B02; Homo sapiens; RNA-Seq
DNA oligonucleotide probes were designed to bind mRNA targets. From 5′ to 3′, each probe is comprised of a 35- to 50-nucleotide target complementary sequence, an ultraviolet (UV) photo cleavable linker and a 66-nucleotide indexing oligonucleotide sequence containing a unique molecular identifier (UMI), RNA ID sequence and primer binding sites. Oligomers from ROIs were disintegrated by UV irradiation, passed through capillaries, pooled into collection plates, and resealed with DAPI fluoromount G mounting medium. Entire slides were imaged at ×20 magnification and morphologic markers were used to select Region Of Interest (ROI) either using circle or organic shapes. Automatic segmentation of ROI based on NeuN marker was used to defined Area of Illumination (AOIs). This allowed to separate Neurons (NeuN+) and cells around. A total of AOIs were exposed to 385 nm light (UV), releasing the indexing oligonucleotides which were collected with a microcapillary and deposited in a 96-well plate for subsequent processing. The indexing oligonucleotides were dried down 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, and unique i5 and i7 sample indices 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. Pooled libraries were single-sequenced at 27 base pairs and with the single-index workflow on an Illumina NovaSeq S4 instrument. FastQ files were converted into DCC files according to manufacturer's pipeline. Digital Count Conversion files were imported back into the GeoMx DSP instrument for QC and data analyses using GeoMx DSP analysis suite version 2.4.2.2 (Nanostring).