alias: E-MTAB-7128_2:Ex1-Day0-3_s
title: Illumina HiSeq 2000 sequencing; Gene expression profiling during human neuronal differentiation
design: Gene expression profiling during human neuronal differentiation
library_construction_protocol: The lt-NES cells (line AF22, derived from iPSCs generated from reprogrammed adult dermal fibroblasts; https://doi.org/10.1371/journal.pone.0029597) were cultured in DMEM/F12+Glutamax (Life Technologies) supplemented with Penicillin (100 U/ml), Streptomycin (100 μg/ml), N2 (1:100, life technologies), B27 (1:1000, Life Technologies) and containing FGF (10 ng/ml; Life Technologies) and EGF (10 ng/ml; Peprotech) in a 5% CO2 incubator. Half of the medium was changed daily and cells were passaged at a ratio of 1:3 upon confluency. Plates were pre-coated using poly-ornithine (0, 1 mg/ml; Sigma) and laminin (2 μg/ml; Sigma, L2020). For spontaneous differentiation, cells were initially plated in complete medium for two days, then medium was changed to medium without growth factors FGF and EGF. After one week, medium was changed to a 1:1 mixture of DMEM/F12+Glutamax and Neurobasal (life technologies) containing N2 (1:200) and B27 (1:100). During differentiation, half of the medium was changed every two to three days containing laminin (1:1000).  RNA was extracted using NucleoSpin RNA kit or NucleoSpin Triprep kit (Macherey-Nagel) according to the supplier's instructions. RNA concentration was measured using Nanodrop ND-1000 and Qubit (Thermo Fisher Scientific). RNA integrity was analysed using Bioanalyzer RNA pico kit (Agilent Technologies). Three biological replicates were collected for each time point in two independent experiments (except for day 14 in experiment 1).  Transcriptome analysis was performed by the STRT RNA-seq method (PMID: 22481528) with minor modifications (PMID: 26874359). Briefly, ten nanogram of high-quality total RNA was converted into cDNA and amplified to form an Illumina-compatible library. ERCC spike-in mixture was diluted 1,000x with water and 1 µl was added to reverse transcriptase master mix before cDNA synthesis. In total, 25 PCR cycles were used; 15 for first, full cDNA amplification and 10 to amplify and introduce sequencing-required motifs.
identifiers.namespace: Department of Biosciences and Nutrition, Karolinska Institutet
identifiers.id: E-MTAB-7128_2:Ex1-Day0-3_s