title: S_425_alpha_S12891
organism: Homo sapiens
description: Protocols: Fibroblasts samples were dissociated with trypsin and centrifuged. Pellets were collected for further processing. All other tissues: cells were lifted with a cell scraper and centrifuged. Pellets were collected for further processing. Fibroblasts: RPMI 1640, FBS 15%, L-Glutamine 1%, Penicillin-Streptomycin 1%; MSC e NSC: as described in Menendez 2013. iPSCs were cultured with mTeSR1 (Stem cell technologies) in feeder-free conditions on hES-qualified Matrigel (BD Biosciences). hESC mTeSR1. as described in Cavallo Troglio 2020. Cortical excitatory neurons were obtained by Ngn2 overexpression in iPSCs. Pluripotent cells were engineered using an all-in-one ePiggyBac (ePB) transposon. We cloned the sequence of Ngn2 - P2A - EGFP - T2A - PuroR 30 sequence into an ePB backbone containing a blasticidin resistance cassette and doxycycline responsive element under the control of hUbC promoter 31. iPSCs were electroporated with the Neon system (ThermoFisher) with the following parameters: 1200V, 30 ms, 1 pulse using 0,5 µg of a helper plasmid expressing a transposase and 4,5 µg of donor plasmid with a transposable element. Electroporated cells were plated in presence of rock inhibitor 5 μM Y-27632 (Sigma). To isolate stably engineered lines, from the day following electroporation, cells were selected by the administration of blasticidin 5µg/mL. Neuronal differentiation was achieved by Ngn2 overexpression, driven by doxycycline administration. Differentiation was performed as already described with minor modifications57. Neuronal maturation was protracted up to five weeks. Stainings on neurons were performed using coverslips nitric acid-treated and coated overnight with poly-L-Lysine at 37°C, following human laminin coating (LN511-02 Biolamina), 3 hours at 4°C. RNeasy Mini Kit - QIAGEN columns truseq stranded total rna with ribozero
attributes.tag: ENA-FIRST-PUBLIC
attributes.value: 2022-12-25
attributes.tag: ENA-LAST-UPDATE
attributes.value: 2022-12-25
attributes.tag: External Id
attributes.value: SAMEA8395569
attributes.tag: INSDC center alias
attributes.value: Istituto Europeo di Oncologia IEO IRCSS S.r.l.
attributes.tag: INSDC center name
attributes.value: Istituto Europeo di Oncologia IEO IRCSS S.r.l.
attributes.tag: INSDC first public
attributes.value: 2022-12-25T00:20:49Z
attributes.tag: INSDC last update
attributes.value: 2022-12-25T00:20:49Z
attributes.tag: INSDC status
attributes.value: public
attributes.tag: Submitter Id
attributes.value: E-MTAB-10244:S_425_alpha_S12891
attributes.tag: broker name
attributes.value: ArrayExpress
attributes.tag: cell_line
attributes.value: KAB4
attributes.tag: cell_type
attributes.value: induced pluripotent stem cell
attributes.tag: common name
attributes.value: human
attributes.tag: disease
attributes.value: Kabuki syndrome
attributes.tag: progenitor cell type
attributes.value: induced pluripotent stem cell
attributes.tag: sample name
attributes.value: E-MTAB-10244:S_425_alpha_S12891
attributes.tag: sex
attributes.value: female