biostudies-arrayexpressQian LiHomo sapienshttps://www.ebi.ac.uk/biostudies/studies/E-MTAB-14086To understand the response of human Hofbauer cells to Listeria monocytogenes infection and how it differs between first trimester and term, we established an infection assay using primary human first-trimester and term Hofbauer cells isolated by fluorescence-activated cell sorting and cultured in vitro for 7 days. We then profiled and compared the transcriptomes of control cells (treated with PBS or Heat-Killed Listeria monocytogenes) and cells infected with Listeria monocytogenes for 8 hours using the NEBNext® Ultra II Directional RNA Library Prep Kit (Illumina).biostudies-arrayexpressNucleic Acid Extraction - For control Hofbauer cells (HBC), cells were lysed in RLT buffer (Qiagen) supplemented with 1% β-ME (Sigma-Aldrich). For Listeria monocytogenes infected HBC, cells were lysed in RLT buffer/β-ME to lyse host cells and centrifuged at 3200 x g for 5 min to pellet out the released intracellular bacteria. Supernatants containing host RNA were collected and stored at -80°C. RNA was extracted using the Single Cell RNA Purification Kit (Norgen) with on-column DNA digestion. Total RNA quality was assessed using the RNA ScreenTape for Tapestation (Agilent).Sample Treatment - Cells were infected with log-phase Listeria monocytogenes at a multiplication of infection (MOI) ~6 and equivalent volumes of PBS or heat-killed Listeria monocytogenes were added to the appropriate wells. Gentamycin was added 1 hour post-infection at 20 μg/ml.Library Construction - Library preparation for bulk RNA-seq was performed using the NEBNext® Ultra II Directional RNA Library Prep Kit for Illumina® and the NEBNext® Multiplex Oligos for Illumina® (96 Unique Dual Index Primer Pairs) in conjunction with NEBNext® Poly(A) mRNA Magnetic Isolation Module as per the manufacturer’s instructions. The Poly(A) mRNA Magnetic Isolation Module was used to extract host mRNA from total RNA of control and infected samples, as Listeria monocytogenes infected samples contain both eukaryotic and prokaryotic RNA. Quality of libraries was assessed on the Agilent D5000 ScreenTape System.Sequencing - Libraries were pooled in equimolar ratios and sequenced at CRUK genomics core (Cambridge) on NovaSeq 6000 S4 lanes (Illumina).Growth Protocol - First-trimester and term Hofbauer cells isolated by fluorescence-activated cell sorting were cultured in 96-, 48- or 24-well plates at a density of 100,000 or 500,000 per well, in ADMEM F12 (Gibco) supplemented with 10% Hi-FBS (Sigma-Aldrich), 10 U/ml Pen/Strep (Gibco), 2.5 μg/ml Amphotericin B (Sigma-Aldrich), 0.5 µg/ml Gentamycin (Sigma-Aldrich), 10 mM L-glutamine (Gibco), 10 mM HEPES (Gibco) and 10 μg/ml M-CSF (Gibco). Media and cytokines were refreshed at day 3 and day 5 of culture and cells were placed in antibiotic-free media 2 days prior to the start of the infection assay.Sample Collection - All tissue samples used were obtained with written informed consent from participants in accordance with the guidelines in The Declaration of Helsinki 2000. First-trimester and term placental tissues were obtained from healthy women with apparent normal pregnancies undergoing medical elective first-trimester terminations (6-13 weeks estimated gestational age (EGA)) and non-labour, elective caesarean sections from uncomplicated pregnancies (36+ weeks EGA) respectively. Ethical approval for the use of first-trimester tissues was obtained from the Cambridge Local Research Ethics Committee (REC 04/Q0108/23), part of the Centre for Trophoblast Research biobank for the ‘Biology of the Human Uterus in Pregnancy and Disease Tissue Bank’ at the University of Cambridge. Overall biobank ethical approval is from the East of England–Cambridge Central Research Ethics Committee (17/EE/0151). Full-term placental tissues were obtained from the Cambridge Blood and Stem Cell Biobank (REC 18/EE/0199).OrganizationMINSEQE ScoreAssays and DataProcessed DataMAGE-TAB FilesData Transformation - Only uniquely mapped reads were considered for the quantification of gene expression based on the human gene annotation (GENCODE v37) using featureCounts in the R package Rsubread (version 2.4.3). This was followed by the calculation of the reads per kilobase per million mapped reads (RPKM) to further normalize the read abundance by the total number of reads and gene length.Sequence Alignment - STAR (version 2.7.8a) was used to map the sequencing reads to the reference human genome (GRCh38_release37_PRI) by requiring the aligned percentage per read > 95%. The adapter sequences were trimmed before the read alignment.MetabolomicsUnknownTranscriptomicsGenomicsProteomicsIllumina NovaSeq 6000RNA-seq of total RNAHomo sapiensNagisa YoshidaQian LiNaomi McGovernfalseBulk RNA-seq profiling the response of human first-trimester and term Hofbauer cells to Listeria monocytogenes infection in vitroTo understand the response of human Hofbauer cells to Listeria monocytogenes infection and how it differs between first trimester and term, we established an infection assay using primary human first-trimester and term Hofbauer cells isolated by fluorescence-activated cell sorting and cultured in vitro for 7 days. We then profiled and compared the transcriptomes of control cells (treated with PBS or Heat-Killed Listeria monocytogenes) and cells infected with Listeria monocytogenes for 8 hours using the NEBNext® Ultra II Directional RNA Library Prep Kit (Illumina).2025-08-04T00:00:00Z2025-08-05T00:02:07.405Z2024-05-17T13:20:50.144ZE-MTAB-14086ERP160355E-MTAB-14087E-MTAB-14088EFO_0002944EFO_0004170EFO_0009653EFO_0003789EFO_0004917EFO_0005518EFO_0003816EFO_0004184EFO_0003969