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Ena-DATASET-POPS-22-03-2019-10:25:08:906-41 - samples

ABSTRACT: Placental biopsies were collected within 30 minutes of birth and flash frozen in RNAlater(ThermoFisher). For each biopsy,total placental RNA was extracted from approximately 5 mg of tissue using the Ã¢Â€ÂœmirVana miRNA Isolation KitÃ¢Â€Â (Ambion) followed by DNase treatment (Ã¢Â€ÂœDNA-free DNA Removal KitÃ¢Â€Â, Ambion). RNA quality was assessed with the Agilent Bioanalyzer and all the samples with RIN values Ã¢Â‰Â¥ 7.0 were used in the downstream experiments. Total RNA-libraries were prepared from 300-500ng of total placental RNA with the Ã¢Â€ÂœTruSeq Stranded Total RNA Library Prep Kit with Ribo-Zero Human/Mouse/RatÃ¢Â€Â(Illumina). Small RNA-libraries were prepared from 150ng of total placental RNA with the Ã¢Â€ÂœNEBNext Multiplex Small RNA Library Prep Kit for IlluminaÃ¢Â€Â(New England Biolabs)and concentrated using the Ã¢Â€ÂœQIAquick PCR purification kitÃ¢Â€Â(Qiagen). Paired libraries were combined and size selected using the Pippin Prep and 3% Agarose Gel Cassette with marker F (Sage Science),pooled and sequenced (single-end, 50bp) using a Single End V4 cluster kit and HiSeq4000 instrument.

PROVIDER: EGAD00001004860 | EGA |

REPOSITORIES: EGA

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Project description:Placental biopsies (n = 64 female placentas, n = 67 male placentas) were selected from healthy pregnancies from the POPs cohort. These patients had no evidence of hypertension at booking and during pregnancy, did not experience pre-eclampsia, Hemolysis, Elevated Liver enzymes, and Low Platelets (HELLP) syndrome, gestational diabetes, or diabetes mellitus type I or type II and other obstetric complications. They delivered live babies with a birth weight percentile in the normal range (20-80th percentile), with no evidence of slowing in fetal growth trajectory. Chorionic villi from the corresponding placentas (free from decidua, visible infarction, calcification, hematoma, or damage) were collected and processed within 30 minutes of separation from the uterus. After repeated washes in chilled phosphate buffered saline, the samples were placed in RNA later (Applied Biosystems) and stored at -80Ã‚Â°C. Total placental RNA was extracted using mirVana Isolation Kit (Ambion). For each placenta, approximately 5 mg of tissue were homogenized in the Lysis/Binding solution for 20 sec at 6 m/s using a bead beater (FastPrep24) and Lysing Matrix D Tubes (MP Biomedicals). The samples were then spun at 13,000 rpm for 5 min at 4Ã‚Â°C and the supernatants recovered. Afterwards, the manufacturer's instructions were followed. Immediately after the RNA extraction, placental RNA samples were DNase-treated using DNA-free DNA Removal Kit (Ambion), aliquoted, and stored in -80Ã‚Â°C. Quantity and quality of the RNA samples were assessed using the Agilent 2100 Bioanalyzer, the Agilent RNA 6000 Nano Kit (Agilent Technologies), and Qubit fluorometer. Libraries were prepared starting with 300-500 ng of good quality total RNA (RIN Ã¢Â‰Â¥7.5) using the TruSeq Stranded Total RNA Library Prep Kit with Ribo-Zero Human/Mouse/Rat (Illumina), according to the manufacturer's instructions. The kit contains 96 uniquely indexed adapter combinations in order to allow pooling of multiple samples prior to sequencing. After determining their size (with the Agilent 2100 Bioanalyzer and the Agilent High Sensitivity DNA Kit by Agilent Technologies) and concentration (by qPCR with the KAPA Illumina ABI Prism Library Quantification Kit, Kapa Biosystems), libraries have been pooled and sequenced (single-end, 125 bp) using a Single End V4 Cluster Kit and an Illumina HiSeq2500 or HiSeq4000 instrument.

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Ena-DATASET-POPS-22-03-2019-10:25:08:906-41 - samples

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