organism: Homo sapiens
description: Protocols: Differentiation into NCSCs was performed as previously described (67), with the exception of NCSCs used in the experiment reported in Fig. 1D-F (16). WBS1, WBS2, WBS3, WBS4, 7dupASD1, AtWBS1 and CTL2 fibroblasts were reprogrammed using the mRNA Reprogramming kit (Stemgent), while the 7dupASD2 and CTL1R lines were reprogrammed with the microRNA Booster kit (Stemgent). The CTL3 line was reprogrammed by transfection with the STEMCCA polycistronic lentiviral vector followed by Cre-mediated excision of the integrated polycistron. 7dupASD3 and CTL4R fibroblasts were reprogrammed using the SimpliconTM RNA Reprogramming kit (Millipore). Prior to differentiation, iPSC lines were cultured on hESC-qualified Matrigel (BD Biosciences) coated plates, diluted 1:40 in DMEM/F-12 and grown in mTeSR-1 medium (StemCell Technologies). They were passaged upon treatment with Accutase (Sigma) and then plated in mTeSR-1 medium supplemented with 5μM Y-27632 (Sigma). Differentiation into NCSCs was performed as previously described (67), with the exception of NCSCs used in the experiment reported in Fig. 1D-F (16). RNA was extracted using the RNeasy Micro Plus kit (Qiagen) according to manufacturer's instructions. Retrotranscribed cDNA was obtained from 0.5-1 μg of total RNA using the SuperScript VILO Retrotranscription kit (Thermo Fisher Scientific). RT-qPCR was performed on a 7500 Fast Real-Time PCR system (Applied Biosystems) using SYBR Green Master Mix (Applied Biosystems) as detecting reagent. A total cDNA amount corresponding to 15 ng of starting RNA was used for each reaction. Each sample was analyzed in triplicate and normalized to GAPDH. Relative mRNA quantity was calculated by the comparative cycle threshold (Ct) method using the formula 2-deltaCt. BAZ1B: CCTCGCAGTAAGAAAGCAAAC (forward); ACTCATCCAGCTCCTTTTGAC (reverse). 4 GAPDH: GCACCGTCAAGGCTGAGAAC (forward); AGGGATCTCGCTCCTGGAA (reverse). NR2F1: AGAAGCTCAAGGCGCTACAC (forward); GGGTACTGGCTCCTCACGTA (reverse). NR2F2: GCAAGTGGAGAAGCTCAAGG (forward); GCTTTCCACATGGGCTACAT (reverse). TFAP2A: GCCTCTCGCTCCTCAGCTCC (forward); CGTTGGCAGCTTTACGTCTCCC (reverse). SOX9: AGTACCCGCACTTGCACAAC (forward); GTAATCCGGGTGGTCCTTCT (reverse). Library preparation for RNA sequencing was performed according to TruSeq Total RNA sample preparation protocol (Illumina), starting from 250 ng - 1 μg of total RNA. cDNA library quality was assessed at an Agilent 2100 Bioanalyzer, using the High sensitivity DNA kit.
attributes.tag: ENA-FIRST-PUBLIC
attributes.value: 2020-02-29T04:05:15Z
attributes.tag: ENA-LAST-UPDATE
attributes.value: 2019-10-21T16:33:18Z
attributes.tag: External Id
attributes.value: SAMEA6104725
attributes.tag: INSDC center name
attributes.value: Department of Oncology and Hemato-Oncology, University of Milan, Italy
attributes.tag: INSDC first public
attributes.value: 2020-02-29T04:05:15Z
attributes.tag: INSDC last update
attributes.value: 2019-10-21T16:33:18Z
attributes.tag: INSDC status
attributes.value: public
attributes.tag: Submitter Id
attributes.value: E-MTAB-8455:AtWBS1.sh1
attributes.tag: broker name
attributes.value: ArrayExpress
attributes.tag: cell type
attributes.value: neural stem cell
attributes.tag: common name
attributes.value: human
attributes.tag: developmental stage
attributes.value: adult
attributes.tag: disease
attributes.value: Williams syndrome
attributes.tag: genotype
attributes.value: wild type genotype
attributes.tag: individual
attributes.value: AtWBS1
attributes.tag: organism part
attributes.value: skin
attributes.tag: progenitor cell type
attributes.value: induced pluripotent stem cell
attributes.tag: sample name
attributes.value: E-MTAB-8455:AtWBS1.sh1
attributes.tag: scientific_name
attributes.value: Homo sapiens