GSM8353482: Lactic14 scMeT-seq; Mus musculus; RNA-Seq
Primary peritoneal macrophages were isolated from 6-month-old male mice C57BL/6. The mice were euthanized, and the peritoneal cavity was injected with 7mL precooled PBS supplemented with 3% fetal bovine serum (FBS, SenBeiJia). The isolation media was drawn back 30 minutes after injection through a 10 mL syringe equipped with a 25 g needle. The isolation media was centrifuged, and erythrocytes were lysed by the addition of RBC lysis buffer (Beyotime). The erythrocyte-free peritoneal cells were resuspended in DMEM media (Gibco) supplemented with 20% FBS, 1mM L-glutamine (Gibco), 1% penicillin-streptomycin (Gibco) and incubated at 37 °C and 5% CO2 for 24 h. After 24 h incubation, non-adherent cells were removed by repeated flushing of the flasks with culture medium to obtain pure peritoneal macrophages. For in vitro treatment, primary mouse macrophages were treatments with 10mM lactate (Sigma-Aldrich),300nM AZD3965 (MedChemExpress), 100uM glycerophosphoinositol (EchelonBioscience), 100uM phosphorylcholine (MedChemExpress), or 80mM Taurine (MedChemExpress) for 72 hours. To evaluate the effects of hypoxia, cells were incubated in normoxic conditions (21% O2) or 3% O2. The scRNA-seq library was constructed following a modified Smart-seq2 protocol(21). In detail, 4ul lysed sample was incubated at 72C for 3 min. The first-strand cDNA was reverse-synthesized using oligo(dT) and template-switching oligonucleotides. The synthesized first-strand cDNAs were amplified by 20 cycles. After purification, 0.1 ng of cDNA was used for Nextera tagmentation (Illumina) and library construction. Sequencing was then performed on Illumina HiSeq 4000.