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Sample GSM4421379

Query DataSets for GSM4421379

Status

Public on Mar 21, 2024

Title

NPC_del_3

Sample type

SRA

Source name

neuronal progenitor cells

Organism

Mus musculus

Characteristics

background strain: C57BL/6N
genotype: 129Sv df/+
developmental stage: neuronal progenitor cells

Growth protocol

Control and mutant cell lines harboring heterozygous deletion (del) or duplication-deletion (dupdel) of 7qF3 locus which is synteny conserved with human 16p11.2 locus were kindly provided by Alea Mills (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, USA). Generation of cell lines described in Horev et al., 2011 (doi: 10.1073/pnas.1114042108). Embryonic stem cells (ESCs) were cultured directly on gelatine-coated plates in M15 medium, supplemented with 15% FBS and 2000 U/ml LIF. Cells were grown for one week before plating them for harvest or for subsequent differentiation. Cells were split every 48 h and media was changed every 24 h. Cell harvest was performed at 70 – 80 % confluency. For generating neuronal precursor cells commited to ventral midbrain lineage or immature dopaminergic neurons, cells were differentiated according to Ferrai et al. (2017). Briefly, the differentiation of ESCs to immature midbrain dopaminergic neurons starts with differentiating ESCs into epiblast stem cells (EpiSCs) by growing them for 4 weeks in N2B27 basal medium containing Activin and FGF2. The EpiSCs were kept in culture for one week, followed by differentiation towards midbrain-specific dopamine neurons. After 5 days of differentiation neuronal progenitor cells were harvested, and after 16 days of differentiation immature dopaminergic neurons were harvested.

Extracted molecule

total RNA

Extraction protocol

Cell lysis was performed on the culture dish, in TRIzol Reagent (Invitrogen, 15596026) at room temperature, and lysate was frozen in liquid nitrogen and stored at -80°C upon processing. Samples were incubated at RT for 5 min and homogenized with 200 ml chloroform per 1 ml of TRIzol, by shaking for 15 s and incubating 3 min at RT. After centrifugation at 12,000xg for 15 min at 4°C, the upper aqueous phase containing the RNA was transferred to a new tube and RNA was precipitated by adding 500 µl HPLC-grade isopropanol, incubating for 10 min at RT, and centrifugation at 12,000xg for 10 min at 4°C. Supernatant was removed and RNA pellet is washed with 75 % ethanol, air-dried for 10 min, resuspended in RNase-free water, and incubated at 55°C for 10 min. DNA was removed by DNase treatment using Turbo DNase (AM2238, Thermo Fisher) according to the manufacturer’s instructions.
RNA-seq libraries were generated from 1 µg of clean RNA using the TruSeq Stranded total RNA library preparation kit according to the manufacturer’s (Sample prep guide 15031048). Samples were sequenced on the NextSeq500, paired end 75 bp, with the NextSeq 500/550 High Output v2 kit (150 cycles), following the manufacturer’s instructions for denaturing, dilution, and sequencing of the libraries.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina NextSeq 500

Description

heterozygous 7qF3 deletion

Data processing

Sequencing reads were mapped to mm9 with STAR v2.6.0c (Dobin et al., 2013), transcripts were quantified with RSEM v1.3.0 (Li and Dewey, 2011), and analysis of count data was performed with DEseq2 v1.22.2 (Love et al., 2014).
Genome_build: mm9

Submission date

Mar 19, 2020

Last update date

Mar 21, 2024

Contact name

Ana Pombo

E-mail(s)

Ana.Pombo@mdc-berlin.de

Organization name

Max Delbrueck Center for Molecular Medecine

Street address

Hannoversche Strasse 28