title: Nuc_16_Rep2
organism: Mus musculus
description: Protocols: Primary cortical neurons were prepared from P0 mouse pups (C57B/6), and cultured as previously described (Kaech, 2006). 106 primary neurons were plated per a Millicell cell culture insert (6-well, Millipore MCRP06H48), coated with poly-D-Lysine and laminin. Separation into soma and neurites was performed using a cotton swab as described (Zappulo, 2017) and further separation of soma on nuclear and cytoplasmic fractions was performed using NE-PER Nuclear and Cytoplasmic Extraction Kit (Thermo 78833) following manufacturer's instructions. Primary cortical were grown in the presence of 200nM 4-thiouridine (s4U) for 24 hours, then s4U was replaced with an excess (20mM) of unmodified uridine (U) and RNA was isolated at the indicated time points, where 0 hours is the time of s4U withdrawal. Isolated RNA was subjected to alkylation by heating to 50°C in the presence of 50 µg/µl Iodoacetamide for 15 min and cleaned up using Ampure beads. RNA was extracted from Trifast using 2-step chloroform extraction and isopropanol precipitation. After alkylation and an additional RNA cleanup step using Ampure beads RNA samples were quantified using qubit and analysed for integrity with tapestation. TrueSeq stranded mRNA Library kit; 1ul of 1:300 ERCC1 spike-in was added to 49ul initial solution with 100ng total RNA.; Final PCR was done with 13-17 cycles' reaction
attributes.tag: ENA-FIRST-PUBLIC
attributes.value: 2023-07-12T00:19:24Z
attributes.tag: ENA-LAST-UPDATE
attributes.value: 2023-07-12T00:19:24Z
attributes.tag: External Id
attributes.value: SAMEA113399633
attributes.tag: INSDC center alias
attributes.value: Berlin Institute for Medical Systems Biology, Max Delbruck Center for Molecular Medicine, Berlin, Germany
attributes.tag: INSDC center name
attributes.value: Berlin Institute for Medical Systems Biology, Max Delbruck Center for Molecular Medicine, Berlin, Germany
attributes.tag: INSDC first public
attributes.value: 2023-07-12T00:19:24Z
attributes.tag: INSDC last update
attributes.value: 2023-07-12T00:19:24Z
attributes.tag: INSDC status
attributes.value: public
attributes.tag: Submitter Id
attributes.value: E-MTAB-12888:Nuc_16_Rep2
attributes.tag: age
attributes.value: 0
attributes.tag: broker name
attributes.value: ArrayExpress
attributes.tag: cell_type
attributes.value: neuron of crebral cortex
attributes.tag: common name
attributes.value: house mouse
attributes.tag: developmental stage
attributes.value: neonate
attributes.tag: fraction
attributes.value: nucleus
attributes.tag: genotype
attributes.value: wild type genotype
attributes.tag: organism part
attributes.value: brain
attributes.tag: sample name
attributes.value: E-MTAB-12888:Nuc_16_Rep2
attributes.tag: scientific_name
attributes.value: Mus musculus
attributes.tag: sex
attributes.value: mixed
attributes.tag: strain
attributes.value: C57BL/6