Name: GSM7796214
Instrument: Illumina NovaSeq 6000
Strategy: RNA-Seq
Source: TRANSCRIPTOMIC
Selection: cDNA
Layout: PAIRED
Construction protocol: For scRNA seq experiements of chorioamniondecidua samples were collected right after delivery, digested, and cell suspension was sorted for live cells. For both human and rhesus bulk RNA seq experiments, amnion was physically separated from chorion and decidua, snap-frozen, and RNA was harvested using Trizol reagent. Bulk RNAseq: Fifteen amnion samples were used (ctrl n=3; LPS n=6; and Adal+LPS n=6). The integrity of purified total RNA from amnion was assessed using the RNA High-Sensitivity Assay on the TapeStation 2200 (Agilent Technologies). 200-300 ng of starting material were used as input material for the NEBNext rRNA Depletion kit (cat# E6350). RNA libraries were then prepared using the NEBNext Ultra II RNA kit (cat# E7765). Two hundred ng of each final library was subject to capture hybridization using Illumina TruSeq Exome, (cat #20020490) using Illumina Exome Panel (cat #20020183) according to the manufacturer's instructions. Quality control for the final libraries was performed using the DNA D1000 Assay (TapeStation 2200 - Agilent Technologies) and quantified using a Qubit dsDNA BR Assay (Life Technologies). Diluted libraries were pooled and sequenced 50 single-end on a HiSeq3000 (Illumina). Single-cell (sc)RNA-seq: For scRNA-seq analysis, eight unseparated chorioamnion-decidua (CAD) samples were used (Ctrl n=2; LPS n=3; and Adal+LPS n=3. Note that 6/8 tissues were also used for bulk RNA-seq analysis (Supplementary Table I). Live-sorted CAD cells were microfluidically partitioned with single cell capture, barcoding, and library construction (10X genomics chromium platform, Pleasanton, California). Single-cell RNA-seq libraries were prepared using the 10X Single Cell 3′ v2 Reagent Kits. Specifically, single cell suspensions were loaded on a Chromium Controller instrument to generate single-cell Gel Bead-In- EMulsions (GEMs). GEM-RT were performed in a Veriti 96-well thermal cycler (Thermo Fisher Scientific, Waltham, MA), following which, GEMs were harvested and the cDNAs were amplified and cleaned up with SPRIselect Reagent Kit. Indexed sequencing libraries were constructed using Chromium Single-Cell 3′ Library Kit for enzymatic fragmentation, end-repair, A-tailing, adapter ligation, ligation cleanup, sample index PCR, and PCR cleanup. The barcoded sequencing libraries were quantified using the KAPA Library Quantification Kit (KAPA Biosystems, Wilmington, MA). Sequencing libraries were loaded on a NovaSeq2 (Illumina, San Diego, CA) with a custom sequencing setting (26bp for Read 1 and 91bp for Read 2).