Bulk RNA-seq of human first-trimester and term Hofbauer cells before and after in vitro culture for 7 days
Bulk RNA-seq of human first-trimester and term Hofbauer cells before and after in vitro culture for 7 days
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-well plates at a density of 50 – 100,000 per well, in 150 µl 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. Freshly isolated cells were immediately lysed in RLT buffer (Qiagen) supplemented with 1% β-ME (Sigma-Aldrich) and stored at -80°C. RNA was extracted using the Single Cell RNA Purification Kit (Norgen) with on-column DNA digestion. Cells collected after in vitro culture for 7 days, were washed gently with warm PBS prior to collection and lysis in RLT buffer/β-ME. Total RNA quality was assessed using the RNA 6000 Pico Kit for Bioanalyzer Systems (Agilent). Library preparation for bulk RNA-seq was performed using the SMARTer® Stranded Total RNA-Seq Kit v3 - Pico Input Mammalian kit (Takara Bio) as per the manufacturer's instructions. Quality of libraries was assessed using the Agilent D5000 ScreenTape System.