| Construction Protocol | Ascl1-iNeurons, grown on a Millicell insert, were treated with cycloheximide (100 g/ml), separated on neurites and soma as described earlier and frozen in liquid nitrogen. 20 inserts were pooled together for neurites isolation, and two inserts for soma isolation. Ribo-seq libraries were generated as previously described (Ingolia, 2014), with some modifications. Each sample was lysed in 1 ml of polysome buffer (20 mM Tris pH 7.4, 150 mM NaCl, 5 mM MgCl2, 1 mM DTT, 1% Triton X-100, 100 ug/ml cycloheximide, 5 U/ml Turbo DNase) and digested with 70 U of RNase I for 40 min at RT. As our analysis revealed that the quality and composition of the libraries generated with and without monosome recovery were comparable (Figure S1), ribosome isolation step was omitted Mouse Embryonic Stem Cells (mESC) with the doxycycline-inducible expression of Ascl1 (Ascl1-mESC) were generated as previously described (Iacovino et al., 2014). mESC were growth on gelatin coated flasks in 80% 2i/20% mESC medium. 2i medium: 50% Advanced DMEM/F12, 50% Neurobasal, 1X N2 (17502048 Life Technologies), 1X B27 (17504044 Life Technologies), 1 mM L-Glutamine (25030024 Gibco), 0.1 mM ß-Mercaptoethanol (ßME), 103 U/mL Leukaemia inhibitory factor (LIF, ESG1107 Merk Millipore), 3µM CHIR99021, 1µM PD03259901 (130-104-170 Milenyi Biotec). mESC medium: KnockoutTM DMEM, 14% Fetal bovine serum (10439016 Life Technologies), 0.1 mM ßME, 1 mM L-Glutamine, 1X MEM Non-essential Amino Acid (11140035 Life Technologies), 1X Nucleosides (ES008D Merck Millipore), 103 U/mL LIF. For neuronal differential into Ascl1-iNeurons and separation on soma and neurites, mESCs were allowed to form embryoid bodies (EB) by growing in suspension in AK differentiation medium (50% Advanced DMEM/F12, 50% Neurobasal, 10% Knockout Serum replacement (10828028 Life Technologies), 1 mM L-Glutamine, 0.1 mM ßME). After 1 day, EB formed from 106 mESCs were plated on a Millicell 6-well cell culture insert (PISP30R48 3 m Millipore), bottom-coated with Matrigel (356237 Corning). Cells were grown in a monolayer differentiation medium (Advanced DMEM/F12 supplemented with B27, N2 and 3 g/ml doxycycline). After 6 days one of the compartments was removed using cotton swabs and the membrane with the remaining compartment (soma or neurites) was used for either RNA extraction with TRIFast (peqGOLD) or protein extraction with 8M UREA, 0.1 M Tris-HCl pH 7.5. After RNAse digest, RNA was isolated using Direct-zol RNA MiniPrep (Biozym) and 400 ng of the isolated footprinted RNA were depleted of rRNA using RiboZero Gold rRNA removal kit (Illumina). The sample was concentrated using RNA clean and concentrator-5 kit (Biozym) and phosphorylated with 10 U PNK for 30 min at 37°C. The RNA was separated on a 15% Urea PAAG, 27-30 nt RNA fragments were eluted from gel Truseq small RNA library prep kit (RS-200-0012 Illumina) |
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