HiSeq X Ten paired end sequencing; RNA sequencing of young mice hippocampus after anesthesia
RNA sequencing of young mice hippocampus after anesthesia
We got the mice from Shanghai SLAC Laboratory Animal (C57BL/J6, Shanghai, China) and separated the mice into three groups randomly. The mice stayed in a temperature and humidity-controlled environment with free access to standard rodent food and water. The mice received sevoflurane anesthesia using the protocol from Lu et al 2017 (PMID: 28968276). Briefly, one group of mice (defined as Group A) received 2.5% sevoflurane with 60% O2 at the flow rate of 2 L/min for 3 min and 1 L/min for 2 h on postnatal day 6, 7, 8, 9 in an anesthetizing chamber (Datex Ohmeda, Madison, USA). Another group of mice (defined as Group B) received 2.5% sevoflurane with 60% O2 at the flow rate of 2 L/min for 3 min and 1 L/min for 2 h on postnatal day 6, 7 in the same anesthetizing chamber. The control group received 60% O2 at the same flow rate and in the same anesthetizing chamber. Polysome fraction was isolated with the method from Lupinacci et al 2019 (PMID: 31052505) with small changes. Briefly, we lysed the hippocampus tissues in a Polytron with 400 uL lysis buffer (using 100 μg/mL cycloheximide), incubated the lysates on ice for 10 min and conducted centrifugation at 10000x g for 10 min at 4°C. The supernatants were collected and loaded into 10–50% sucrose gradients. Gradients were placed in a Beckman SW41Ti rotor and centrifuged at 39,000 rpm at 4 °C for 2.5 h, after which the polysome fractions were collected. Then, polysome-associated RNA was extracted with TRIZOL according to the manufacturer's protocol (Invitrogen, Waltham, USA). Total RNA was extracted directly from the hippocampus tissues following the same TRIZOL protocol. The extracted RNA was treated with DNase to remove genomic DNA contamination. Isolation of mRNA was based on NEBNext PolyA mRNA Magnetic Isolation Module (New England Biolabs, Ipswich, USA) and the mRNA was further used with NEB Next Ultra Directional RNA Library Prep Kit for Illumina (New England Biolabs, Ipswich, USA) for sequencing library preparation.