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Sample GSM1612340

Query DataSets for GSM1612340

Status

Public on Jul 03, 2017

Title

Wildtype differentiating Neural Stem Cell RNAseq-2

Sample type

SRA

Source name

Neural stem cells

Organism

Mus musculus

Characteristics

genotype: Wildtype

Growth protocol

Briefly, forebrains without the olfactory bulb (4 mice/genotype, age- and sex-matched) were dissociated mechanically, followed by enzymatic digestion using PPD (2.5 units/ml papain, 1 unit/ml DNase I, 200 mg/100 ml Dispase II) in Dulbecco's modified Eagle's medium high glucose (Cellgro). After filtering through a 70-μm cell strainer (catalog number 252350; BD Falcon), the single cell suspension was loaded onto 50% Percoll (Sigma). The NSPCs were separated from other cells by ultracentrifugation at 12,700 rpm for 30 min at 20 °C using an SW41 Rotor (Beckman, CA). The fraction containing NSPCs (immediately above the red blood cell layer in the gradient) was collected, washed with phosphate-buffered saline, and cultured with Dulbecco's modified Eagle's medium/F-12 medium containing 20 ng/ml FGF-2 (catalog number K1606; Peprotech), 20 ng/ml epidermal growth factor (catalog number A2306; Peprotech), 1% N2 supplement (catalog number 17502-048; Gibco), 1% antibiotic-antimycotic (catalog number 15240-062; Gibco), and 2 mM L-glutamine (catalog number 25030-081; Gibco) in a 5% CO2 incubator at 37 °C. Half of the medium was replaced every 2–3 days.

Extracted molecule

total RNA

Extraction protocol

Five to ten million NSCs were treated with 1% formaldehyde for 10 min at room temperature with gentle shaking. Fixation was terminated by adding 2M Glycine to reach 0.125M final concentration, and continue shaking for additional 5min. Cells were collected by cell scrapers and spin down at 1500 rpm for 10 min. Cell pellet was resuspended in 1ml Nuclei Swelling Buffer (10 mM Hepes/pH7.9, 0.5% NP-40, 1.5 mM MgCl2, 10 mM KCl, 0.5 mM DTT and protease inhibitor cocktail), incubated on ice for 10 min and centrifuged at 5000 rpm for 5 min. Nuclear pellets were further lysed in 1ml SDS lysis buffer (20 mM Hepes/pH7.9, 25% glycerol, 0.5% NP-40, 0.5% Triton X-100, 0.42 M NaCl, 1.5 mM MgCl2, 0.2 mM EDTA and protease inhibitor cocktail). Cell lysate were sonicated 5 times with 6-9W output to obtain DNA fragments between 200-500bp. After sonication, nuclear lysate was cleared by centrifugation at 13000 rpm for 10 min to keep supernatant. The nuclear lysate was diluted with 3-4 volumes of dilution buffer (0.01% SDS, 1% Triton X-100, 1.2 mM EDTA, 167 mM NaCl, 16.7 mM Tris-HCl/pH8.0 and protease inhibitor cocktail). Immunoprecipitation was performed with antibodies specific to Foxo3a (1:1000, Bethyl) rotating overnight at 4ºC. 20 μl Protein-G Dynabeads (Life technologies) were added for additional 2 hours. TSE I (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 150 mM NaCl, 20 mM Tris-HCl/pH 8.1), TSE II (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 500 mM NaCl, 20 mM Tris-HCl/pH 8.1), TSE III (0.25 M LiCl, 1% NP-40, 1% deoxycholate, 1 mM EDTA, 10 mM Tris-HCl/pH 8.1), and washed twice with TE buffer before eluted with 1% SDS and 0.1 M NaHCO3. The elution was incubated at 65 ºC for at least 6 hours to reverse the formaldehyde cross-linking, then DNA fragments were purified by using PCR Purification Kit (Qiagen). ChIPed DNA was subjected to library preparation. For genomic DNA extraction, Genomic DNA was first sonicated to 100-500bp, and mixed with 100 ml solution containing 50 mM HEPES buffer (pH 7.9), 25 mM MgCl2, 250 mM UDP-6-N3-Glu and 2.25 mM wild-type b-glucosyltransferase. Reactions were incubated for 1 h at 37°C. DNA substrates were purified via Qiagen DNA purification kit. 150 mM dibenzocyclooctyne modified biotin was then added to the purified DNA, and the labeling re- action was performed for 2 h at 37°C. The biotin-labeled DNA was enriched by Streptavidin-coupled Dynabeads (Dynabeadsw MyOneTM Streptavidin T1, Life Technologies) and purified by Qiagen DNA purification kit for library preparation. RNA isolation was done by Trizol.
25ng of input genomic DNA or experimental enriched DNA were utilized for each library construction. 150-300 bp DNA fragments were selected by AMPure XP Beads (Beckman Coulter) after the adapter ligation. An Agilent 2100 BioAnalyzer was used to quantify the amplified DNA, qPCR was applied to accurately quantify the library concentration. 20 pM diluted libraries were used for sequencing. 50-cycle single-end sequencings were performed using Illumina HISeq 2000. Image processing and sequence extraction were done using the standard Illumina Pipeline. RNA-seq libraries were generated from duplicated samples per condition using the Illumina TruSeq RNA Sample Preparation Kit v2 following manufacturer’s protocol. The RNA-seq libraries were sequenced as 50-cycle pair-end runs using Illumina HiSeq 2000.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiSeq 2000

Description

RNA-seq

Data processing

Basecalls performed using CASAVA version 1.4
ChIP-seq reads were aligned to the mm9 genome assembly using bowtie version 0.12.9 with the standard parameters.
RNA-seq reads were aligned using tophat v2.0.8 and differential RPKM expression values were extracted using cuffdiff v2.2.1
Peaks were called using MACS version 1.4.2 with the following setting: BW (200), GSIZE (mm).
Genome_build: mm9

Submission date

Feb 17, 2015

Last update date

May 15, 2019

Contact name

Bing Yao

E-mail(s)

bing.yao@emory.edu

Phone

4047271725

Organization name

Emory University