title: GSM7136276: TMA2_20221115 | 012 | Liver cells; Homo sapiens; RNA-Seq
library_construction_protocol: Three FFPE tissue sections of 5-µm from liver biopsy tissue microarray (TMA) with VILI (n=6) and AIH (n=8) were mounted on positively charged histology slides. Sections were incubated at 65 °C for 1 hour. Then, sections were deparaffinized in 3 xylol baths of 5 minutes and rehydrated in ethanol gradient from 100% EtOH 2 baths of 5 minutes, followed by 95% EtOH 5 minutes. Slides were then washed in 1X PBS. Antigen retrieval was carried out with Tris-EDTA pH 9.0 buffer at 100°C for 20 minutes at low pressure. Slides were first immersed into hot water for 10 seconds and then immersed into Tris-EDTA buffer. The cooker vent kept open during the procedure to ensure low pressure and it was allowed to reach 100°C. Slides were then washed with 1X PBS, and incubated in proteinase K containing PBS (1ug/ml) for 15 minutes at 37°C and washed again in 1X PBS. Tissue were post-fixed in 10% neutral-buffered formalin (NBF) for 5 minutes, washed two times 5 minutes in NBF stop buffer (0.1M Tris Base, 0.1M Glycine) and finally one time in 1X PBS. The mix of Whole Transcriptome Atlas probes (WTA, Nanostring) was dropped on each section and covered with HybriSlip Hybridization Covers. Slides were then incubated for hybridization overnight at 37°C in a Hyb EZ II hybridization oven (Advanced cell Diagnostics). The day after, HybriSlip covers were gently removed and 25 minutes stringent washes were performed twice in 50% formamide and 2X saline sodium citrate (SSC) at 37 °C. Tissues were washed for 5 min in 2× SSC, then blocked in Buffer W (Nanostring Technologies) for 30 min at room temperature in a humidity chamber. Next, 500 nM Syto83 and antibodies targeting CD3 (BioRad, clone CD3-12), CD20 (Novus, clone IGEL/773) and Arginase (Cell Signaling, clone D4E3M) in Buffer W were applied to each section for 1 h at room temperature. Slides were washed twice in fresh 2× SSC then loaded on the GeoMx DSP.  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 Arginase+ markers were used to defined Area of Illumination (AOIs). This allowed to separate liver cells (Arginase+) and cells around liver. A total of 59 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.