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Sample GSM1369005

Query DataSets for GSM1369005

Status

Public on Apr 15, 2014

Title

NPC_Time_11_rep0

Sample type

SRA

Source name

H1-derived neural progenitor cells

Organism

Homo sapiens

Characteristics

cell line: H1
cell type: ES-derived neural progenitor cells
time: Day 11

Treatment protocol

N/A

Growth protocol

H1 human neural stem cells were prepared as described previously (Arnold et al., 2008; Goff et al., 2009) and grown at 37°C, 5% CO2 on 1:4 diluted Matrigel-coated wells in neural proliferation medium (NPM; 50% DMEM/F12 Glutamax, 50% Neurobasal medium, 0.5X N2, 0.5X B27 without vitamin A, 20 ng/ml FGF [Life Technologies]). For differentiation, cells were plated at a density of 106 cells per well in a 6-well plate and allowed to proliferate for one day in the NPM medium. Neural induction was then initiated by withdrawal of FGF and addition of BDNF by switching the medium to neural differentiation medium (NDM; 100% Neurobasal medium, 1X B27 without vitamin A [Life Technologies], 10 ng/ml BDNF [Peprotech, Rocky Hill, NJ].) Differentiating cultures were maintained by refreshing NDM every other day until collection. Samples of these cultures were collected at days 0, 1, 2, and 4. Remaining cells (those designated for collection at days 5, 11, and 18) were replated at day 4 at a density 106 cells per well of a Poly-D-lysine/laminin-coated 6-well plate.

Extracted molecule

polyA RNA

Extraction protocol

Cells were harvested with Accutase (Stem Cell Technologies, Vancouver, Canada). Total RNA was isolated using TRIzol (Life Technology, Carlsbad, CA)/chloroform extraction followed by spin-column purification (RNeasy mini kit, Qiagen, Venlo, Netherlands) according to the manufacturer instructions. RNA concentration and purity were determined using a Nanodrop (Thermo Fisher, Waltham, MA). RNA integrity was assessed on a Bioanalyzer (Agilent, Santa Clara, CA) using the RNA 6000 RNA chip. High-quality RNA samples (RNA Integrity Number ≥8) were used for library preparation.
200ng total RNA was used as input for the TruSeq RNA-Seq kit (Illumina) for each of the samples described. Prior to sequencing, libraries were run on a Bioanalyzer DNA7500 chip to assess purity, fragment size, and concentration. Libraries free of adapter dimers and with a peak region area (220–500 bp) ≥80% of the total area were sequenced. Individually barcoded samples were pooled and sequenced on the Illumina HiSeq 2000 platform.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiSeq 2000

Data processing

Reads were aligned to the human (hg19) reference genome assemblies using Tophat2 with default options.
Aligned fragments were used as input for Cuffdiff2 to determine expression levels (FPKM) and differential expression between conditions using the default options.
CummeRbund v2.1 (http://compbio.mit.edu/cummeRbund/) was used to process, index, and visualize the output of the Cuffdiff2 analyses.
Genome_build: hg19
Supplementary_files_format_and_content: CummeRbund diffTable output. Contains log ratios and differential significance test results.
Supplementary_files_format_and_content: see diffTable output for locations on the genome

Submission date

Apr 14, 2014

Last update date

May 15, 2019

Contact name

Loyal A Goff

E-mail(s)

loyalgoff@jhmi.edu

Organization name

Johns Hopkins University

Department

Institute of Genetic Medicine