Lmd7000 laser capture microdissection system

Tissue samples from human or mice were cut into 5–10 consecutive sections (8 μm) and the epithelial layer contained 30–50 cells on each section was micro-dissected with a Leica LMD7000 laser-capture microdissection system. RNA was isolated from the mini-bulk samples and cDNA was prepared for sequencing based on the Geo-seq protocol^{59 (link)}. Sequencing libraries were built using the TruePrep DNA Library Prep Kit V2 for Illumina (Vazyme), and evaluated by Bioanalyzer (DNA HS kit, Agilent). RNA-seq data were mapped to GRCh38 human genome and GRCm38 murine genome by HISAT2 (version 2.1.0)^{60 (link)} with default parameter for human and murine samples, respectively. The gene expression matrix of raw reads counts after annotation by HTSeq (version 0.6.1p1) was processed using the DESeq2 (version 1.22.2)^{61 (link)} and visualized by showing the first 3 dimensions calculated by plotPCA function. We used TRIZOL to extract bulk RNA from patient biopsy (n = 45), and constructed sequencing library using NEBNext Ultra II RNA Library Prep Kit for Illumina. Sequencing data were processed by HISAT2 and HTseq as described above. The normalized expression from bulk samples of human precancerous lesions was used to estimated neutrophil fraction with CIBERSORT (version 1.06)^{62 (link)}.

Immunofluorescence of PNPLA9 in human or mouse placental sections was performed as previously described (102 (link)). A similar procedure was used for detection of CGI58, including blocking in donkey serum for 2 hours and incubation with primary anti-CGI58 rabbit monoclonal antibody and a secondary antibody, as detailed in Supplemental Table 4. For laser-capture microdissection, frozen sections (7 μm) of cryomold-embedded mouse placentas were transferred to polyethylene naphthalate membrane slides (Thermo Fisher Scientific) and stained with toluidine blue. The Leica LMD7000 laser-capture microdissection system was used to visualize, circumscribe, and collect 3 tissue types: labyrinth, junctional zone, and decidua. RNA was extracted from the microdissections, using the RNeasy FFPE Kit (QIAGEN, catalog 73504) according to the manufacturer’s instructions. The SuperScript VILO synthesis kit enzyme (Thermo Fisher Scientific, catalog 11755500) was used for the reverse transcription reaction, according to the manufacturer’s instructions. qPCR was performed in duplicate as detailed above.