Allprep dna rna ffpe tissue kit

Each FFPE tissue was cut into five, 10 micron scrolls and DNA isolated using the AllPrep DNA/RNA FFPE Tissue kit per manufacturer’s instruction (Qiagen, Valencia, CA). Concentration and purity of isolated DNA was determined by NanoDrop, while RNA quality was assessed using a Bioanalyzer (Agilent Technologies, Santa Clara, CA). The extracted DNA was used for deep sequencing of the TCR-β chain of all T cells in placental villi using the ImmunoSEQ platform v4 (Adaptive Biotechnologies, Seattle, WA). Investigators running this assay were blinded to the group allocation of the samples. Briefly, the assay amplifies rearranged complementarity-determining region 3 (CDR3) of TCRs using a bias-controlled, multiplex-PCR method (23 (link), 24 (link)). Samples were sequenced on an Illumina MiSeq platform using a 100 cycle paired end protocol (Illumina, San Diego, CA). The quantity of nucleated cells and T cells was determined during sequencing through the addition of reference gene primers and TCR-β specific primers (24 (link)). Raw sequencing data were uploaded and are freely accessible through immuneACCESS (clients.adaptivebiotech.com/pub/enninga-2020-ji; DOI 10.21417/EALE2020JI).