title: Fever-range temperature alters continual efferocytosis mediated by mouse pro-inflammatory macrophages
design: Fever-range temperature alters continual efferocytosis mediated by mouse pro-inflammatory macrophages
library_name: Sample 7_p
library_construction_protocol: Adult male C57BL/6 Lys5.1 mice (Charles River) were used to generate BMDM. After euthanasia, femurs and tibias were recovered and were put in Roswell Park Memorial Institute medium (RPMI, 72400, Gibco) containing 1% penicillin/streptomycin (P/S, CABPES01-0U, Eurobio). Bone marrow cells were flushed from bones, and then separated with a 25-gauge needle. After centrifugation, red cells were lysed using a hypotonic solution (150 mM NH4Cl, 0.1 mM EDTA disodium salt and 1 mM KHCO3). After bone marrow cell centrifugation, cells pellets were suspended in complete Iscove's Modified Dulbecco's Medium (IMDM, L0190, Biowest) supplemented with 1% P/S, 1X non-essential amino acids (NEAA, M7145, Sigma-Aldrich), 1 mM sodium pyruvate (S8636, Sigma-Aldrich) and 10% foetal bovine serum (FBS, 10270106, Gibco) and passed through a filter with 100 μm pores (130-098-463, Miltenyi Biotec). Bone marrow cells were concentrated at 1 millions cells/ml in complete IMDM supplemented with 10 ng/ml of M-CSF (130-101-706, Miltenyi Biotec) and cultured at 37°C and 5% CO2 during 7 days. At day 7, medium was removed and replaced with a pro-inflammatory polarisation medium consisting in complete IMDM with 100 ng/ml of LPS (Escherichia coli O55:B5, L6529, Sigma-Aldrich) and 50 ng/ml of IFN-γ (130-105-774, Miltenyi Biotec) for two days. Pro-inflammatory polarisation medium were replaced with RPMI medium containing 1% P/S, 1% NEAA, 1% sodium pyruvate and 10% FBS. Cells were placed at 37°C or at 39.5°C under 5% CO2 during 4 and 8 hours. Total RNA from pro-inflammatory macrophages cultured at 37°C or at 39.5°C for 4, or 8 hours was extracted with RNeasy© mini kit (74106, Qiagen) according to the manufacturer's recommendations. DNA were eliminated with DNase I (79254, Qiagen) during extraction. Purity of mRNA was assessed by determining the ratio of absorbance readings at 260 nm and 280 nm, and sample quality was verified with the NanoDrop 2000 spectrophotometer (ThermoFisher scientific). The stability of mRNA was determined with TapeStation 4150 (Agilent) with RNA ScreenTape kits (Agilent). Library construction and sequencing were performed by the Beijing Genomics Institute (BGI, Shenzhen, China).
identifiers.namespace: European Bioinformatics Institute
identifiers.id: E-MTAB-14408:Sample 7_p