GSM5222720: APP-6m_3; Mus musculus; RNA-Seq
Left and right hippocampus tissue was homogenized in 1.0mL Trizol reagent: 15 minute incubation and subsequent grinding using a pestle until nothing but insoluble connective tissue remained. The homogenized mix was pipetted up and down and the solution was stored at -80°C. 0.5mL of homogenized mixture was phase separate by the addition of 200mL of chloroform (Sigma, C2432) and mixed by inversion, incubated for three minutes and centrifuged at 12 500xg for fifteen minutes at 4°C. The top (aqueous) layer was transferred to a new tube and mixed with 500mL of isopropanol (Fisher, 67-63-0), followed by a 1 hour incubation at -20°C and centrifugation at 12 500xg for ten minutes at 4°C. The supernatant was removed and the pellet was washed and mixed with 1mL of 75% ethanol, followed by a centrifugation at 7 600xg for five minutes at 4°C. The supernatant was removed and the pellet was allowed to dry for 1 minute before eluting in 50mL of nuclease free H2O. The eluted RNA was heated at 55°C for fifteen minutes, and subsequently incubated with 1mL of DNaseI (NEB, M0303), 10mL of 10x DNaseI buffer (NEB, B0303), and 39mL of nuclease free H2O for 15 minutes at 37°C. The RNA was further cleaned using the Zymo Research RNA clean and concentrator kit -25 (R1017). The RNA was stored at -80°C. 1.5ug total RNA was size selected using the Invitrogen MirVana kit (AM1561). 90uL of the short elution was polyadenylated using 12uL 10x polyA polymerase buffer (NEB, B0276), 12uL 10mM ATP (NEB, B0756A), 6uL polyA polymerase (NEB, M0276) and incubated at 37C for 30min. The polyadenylated short fraction and the long fraction were combined and purified and concentrated using the RNeasy MinElute kit (Qiagen, 74204). 12uL of the RNA was adaptor ligated using 4uL NEBNext Quick ligation buffer (B6058), 0.66uL RNA CS (Nanopore), 1.3uL RTA adaptor (Nanopore), 2uL 2 000 000U T4 DNA ligase (NEB, M0202). The reaction was incubated at room temperature for 10 minutes. The sample was subsequently reverse transcribed using 1.5uL of the Lucigen OmniAmp polymerase (F831942-1), 10uL 10x OmniAmp buffer (Lucigen, F883707-1), 8uL dNTP (NEB, N0447), 6uL 100mM MgSO4 (Lucigen, F98695-1), 10uL Betaine (Lucigen, F881901-1), 5uL random primer mix (NEB, S1330), and 39.5uL RNase free H2O. The mixture was incubated for 50C – 10min, 70C – 2min. Samples were cleaned by 2.88x Omega Mag-BindÒ TotalPure NGS beads. The 20uL library was ligated to the Nanopore RMX adaptor using 6uL RMX adaptor (Nanopore), 8uL NEBNext quick ligation buffer, 3uL RNase free H­2O, and 3uL 2 000 000U T4 DNA ligase and incubated for 10 minutes at room temperature. The adapted library was 1x bead cleaned using the Omega Mag-BindÒ TotalPure NGS beads, eluted in Nanopore elution buffer. Library was loaded onto Nanopore PromethION according to manufacturer's instructions.
GEO
GSM5222720