Bulk RNA-seq profiling the response of human first-trimester and term Hofbauer cells to Listeria monocytogenes infection in vitro
Bulk RNA-seq profiling the response of human first-trimester and term Hofbauer cells to Listeria monocytogenes infection in vitro
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). 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. 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. 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). 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.