GSM8733709: miRNA-seq of motor neuron axons treated with primary muscle EVs, rep 3; Mus musculus; miRNA-Seq
RNA extraction MN axonal RNA was extracted from outer compartment of radial MFCs at 14DIV. Axonal RNA was extracted by removing the PBS (from prior wash) from the outer compartment and adding 100µL Qiazol lysis reagent (Qiagen). Inner well was filled with higher volume of PBS to disable the inward flow of lysis reagent towards the inner (soma) compartment and prevent soma contamination. Axons washed off the plate by pipetting the Qiazol around the outer well for 30 seconds. RNA from somata in the inner compartment was extracted with 100µL Qiazol reagent, and lysate was collected in a similar manner. cDNA for axon and soma was prepared with High-Capacity Reverse Transcription Kit (Thermo; Cat. 4368814). Exosomal RNA was extracted by dissolving the exosomal pellet in 700µL Qiazol. RNA was extracted using the RNAeasy micro kit (Qiagen) according to manufacturer's protocols and RNA yield was quantified using Qubit fluorometer Qubit fluorometer (RNA broad range assay kit; ThermoFisher Scientific). For standard whole-cell RNA preparations from MN / muscle cultures, cultures were lysed in 700µL TRI reagent, and RNA was isolated with ordinary chloroform, isopropanol precipitation protocol. small RNA libraries were constructed using the QIAseq miRNA library kit following manufacturer's instructions. mRNA libraries were constructed with the MARS-seq protocol (Keren-Shaul et al., Nat. Prot. 2019)