GSM6454679: 4wkM28Ddit3-Het5-tdtomato; Mus musculus; RNA-Seq
For experiments on OSN developmental populations (INPs, iOSNs, mOSNs), cells were sorted across multiple days, pelleted (10 minutes at 800g) and frozen at -80 until the day of extraction. Lysates were prepared from roughly 1e6 cells per condition using a lysis buffer (20mM Tris pH=7.5, 150mM NaCl, 5mM MgCl2, 1mM DTT, 100ug/mL cycloheximide, 1%(v/v) Triton X-100 and 25U/mL TURBO DNase), RNA was quantified by Quant-iT RiboGreen assay (Thermo Fisher) and lysate containing 100ng of RNA was mixed with 300uL of Trizol (Invitrogen). In all other experiments, 10-20k cells were sorted directly into 500uL of Trizol LS (Sigma). RNA was recovered from Trizol using the Direct-Zol RNA Miniprep kit (Zymo), precipitated overnight with 300mM sodium acetate and 1.5 volumes of isopropanol, digested for 30min at 37°C with TURBO DNase (Invitrogen) and cleaned up on 1.8x Ampure Beads (Beckman Coulter). <10ng RNA was used for library prep, as measured by Quant-iT RiboGreen assay. Libraries were prepared using the SMARTER Stranded Total RNA-seq Kit v2 - Pico Input Mammalian (Takara Bio) and the manufacturer's instructions.