RNA-seq analysis of AS-NMD targets in developing mouse neurons
RNA-seq analysis of AS-NMD targets in developing mouse neurons
We analyzed undifferentiated TRE-Ngn2 mouse embryonic stem cell (ESCs) containing a doxycycline (Dox) inducible Ngn2 transgene (day 0) and TRE-Ngn2 cells undergoing neuronal differentiation in response to Dox-induced Ngn2 expression (days 3 to 24). Undifferentiated TRE-Ngn2 ESCs were propagated in gelatinized 6-well plates (Thermo Fisher Scientific Nunc™ cat# 140675) in 2i+LIF medium, as described [PMID: 30914406]. We adapted published protocols for Ngn2-induced glutamatergic neuronal differentiation [PMIDs: 22719915 and 34971564]. On the day of Ngn2 induction, 6-well plates were coated with Geltrex (Gibco, cat# A1413302), diluted 1:50 in cold DMED/F12, for 2 hours in a humidified incubator at 37°C, 5% CO2. Geltrex was aspirated and TRE-Ngn2 ESCs were immediately plated in the iN-D0 medium containing a 1:1 mixture of Neurobasal (Thermo Fisher Scientific, cat# 21103049) and DMEM/F12 (Sigma, cat# D6421) supplemented with 100 units/ml PenStrep (Thermo Fisher Scientific, cat# 15140122), 1×N2 (Gibco, cat# 17502048), 1×B27 with retinoic acid (Gibco, cat# 17504044), 1 µg/ml laminin (Sigma, cat# L2020), 20 µg/ml insulin (Sigma, cat# I0516), 500 µM L-glutamine (Gibco, cat# 25030-024), 2 mM db-cAMP (Sigma, cat# D0627), 10 ng/ml NT-3 (Miltenyi Biotec, cat# 130-093-973), 100 µM 2-mercaptoethanol and 2 μg/ml Dox. The cells attached to the wells were then cultured in a humidified incubator at 37°C, 5% CO2. On day 2 (i.e. 48 hours later), the medium was changed completely to the iN-D2 medium similar to iN-D0 except containing 200 µM L-ascorbic acid (Sigma, A4403) instead of 2-mercaptoethanol and 1 μg/ml instead of 2 μg/ml Dox. Starting from day 4, we replaced only ~50% of conditioned medium with fresh Dox-free medium. The iN-D4 medium used for this purpose on day 4 had the same composition as iN-D2, but lacked Dox. The half-volume changes on day 6 onwards were done using the iN-D6 medium containing Neurobasal supplemented with 1×B27 with retinoic acid, 1×CultureOne supplement (Gibco, cat# 15674028), 500 µM L-glutamine, 1 µg/ml laminin, 100 units/ml PenStrep, 200 µM L-ascorbic acid, 10 ng/ml NT-3, 10 ng/ml BDNF (Miltenyi Biotec, cat# 130-093-811), and 2 mM db-cAMP. Day 0 non-induced samples were produced by plating TRE-Ngn2 ESCs in 2i+LIF at 1×10^5 per well and then harvesting RNA 3 days later. A similar approach was used to prepare undifferentiated cultures of wild-type IB10 ESCs (see below). We plated 6-8×10^4 TRE-Ngn2 ESCs per well to produce day 3, 6, 12 and 24 differentiated samples. Where indicated, samples were treated with either DMSO or 100 µg/ml cycloheximide (CHX) for 6 h prior to cell lysis.To identify natural AS-NMD targets, cultures of wild-type (IB10) ESCs, neural progenitor cell (NPC) neurospheres, and primary neurons were treated with either DMSO or 100 µg/ml CHX for 6 h at 37°C. Alternatively, we prepared nuclear and cytoplasmic fractions from untreated cells using the PARIS kit (Thermo Fisher Scientific, cat# AM1921). Primary NPC cultures were prepared by dissecting embryonic day 14.5 (E14.5) mouse embryonic cortices in Hank's Balanced Salt Solution (1×HBSS, Thermo Fisher Scientific; cat# 14025092) and dissociating them by trituration. The NSCs were then cultured as neurospheres in reconstituted NeuroCult Proliferation Kit medium (STEMCELL Technologies, cat# 05702) supplemented with 20 ng/ml recombinant human EGF (STEMCELL Technologies, cat# 78006). For passaging, neurospheres were dissociated with the NeuroCult Chemical Dissociation Kit (STEMCELL Technologies, cat# 05707) as recommended. Primary cortical neurons were isolated from E15.5 mouse embryos and cultured as described [PMID: 17406484]. Briefly, cortices were dissociated with 2.5% trypsin (Thermo Fisher Scientific, cat# 15090046) and plated on poly-L-lysine (Sigma, cat# P2636) coated 6-well plates or 18-mm round coverslips in Minimum Essential Media (MEM) with L-glutamine (Thermo Fisher Scientific, cat# 11095080), 0.6% glucose (Fisher Scientific, cat# 10335850) and 10% horse serum (GIBCO/Life Technologies) at the density of 1×10^6 neurons per well and 2.5×10^4 neurons per coverslip. Neurons in 6-well plates were cultured for 5 days without glial feeders, whereas neurons attached to coverslips were transferred to wells containing a monolayer of newborn rat astrocytes [PMID: 17406484], and cultured in neuronal maintenance medium [MEM with L-glutamine, 0.6% glucose and 1× Neurocult SM1 neuronal supplement (STEMCELL Technologies, cat# 05711)] for 21 days replacing half of the medium every 3-4 days. The TRE-Ngn2 samples were lysed using TRIzol Reagent (Thermo Fisher Scientific, cat# 15596026). The lysates were immediately frozen and stored at -80°C and processed on the same day using the TRIzol Plus RNA Purification Kit (Thermo Fisher Scientific cat# 12183555). RNAs were eluted in nuclease-free water (Invitrogen, cat# AM9939). RNAs form the natural samples were purified using either the Purelink RNA mini kit (Thermo Fisher Scientific, cat#12183025; IB10 ESCs and NPCs) or the RNeasy micro kit (Qiagen, cat#74004; primary neurons). RNA concentration was estimated using Nanodrop and measured with the Qubit RNA BR Assay Kit (Fisher Scientific #Q10211). Additionally, RNA quality was evaluated using the Agilent Technologies RNA ScreenTape Assay (5067-5576 and 5067-5577). For the TRE-Ngn2 time series, intact poly(A) RNA was purified from total RNA samples (100-500 ng) with oligo(dT) magnetic beads and stranded mRNA sequencing libraries were prepared as described using the Illumina TruSeq Stranded mRNA Library Prep kit (20020595) and TruSeq RNA UD Indexes (20022371). Purified libraries were qualified on an Agilent Technologies 2200 TapeStation using a D1000 ScreenTape assay (cat# 5067-5582 and 5067-5583). The molarity of adapter-modified molecules was defined by quantitative PCR using the Kapa Biosystems Kapa Library Quant Kit (cat#KK4824). Individual libraries were normalized to 1.30 nM in preparation for Illumina sequence analysis. Sequencing libraries were chemically denatured and applied to an Illumina NovaSeq flow cell using the NovaSeq XP chemistry workflow (20021664). Following transfer of the flowcell to an Illumina NovaSeq instrument, a 2 x 51 cycle paired end sequence run was performed using a NovaSeq S1 reagent Kit (20027465).  For the natural samples, intact poly(A) RNA was purified from total RNA (100-500 ng) with oligo(dT) magnetic beads and stranded mRNA sequencing libraries were prepared as described using the Illumina TruSeq Stranded mRNA Library Preparation Kit (RS-122-2101, RS-122-2102). Purified libraries were qualified on an Agilent Technologies 2200 TapeStation using a D1000 ScreenTape assay (cat# 5067-5582 and 5067-5583). The molarity of adapter-modified molecules was defined by quantitative PCR using the Kapa Biosystems Kapa Library Quant Kit (cat#KK4824). Individual libraries were normalized to 5 nM and equal volumes were pooled in preparation for Illumina sequence analysis. Sequencing libraries (25 pM) were chemically denatured and applied to an Illumina HiSeq v4 single read flow cell using an Illumina cBot. Hybridized molecules were clonally amplified and annealed to sequencing primers with reagents from an Illumina HiSeq SR Cluster Kit v4-cBot (GD-401-4001). Following transfer of the flowcell to an Illumina HiSeq 2500 instrument (HCSv2.2.38 and RTA v1.18.61), a 50 cycle single-read sequence run was performed using HiSeq SBS Kit v4 sequencing reagents (FC-401-4002).