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Sample GSM521652

Query DataSets for GSM521652

Status

Public on May 01, 2010

Title

NPC_RNASeq

Sample type

SRA

Source name

Neural progenitor cells

Organism

Mus musculus

Characteristics

strain: C57BL/6
cell type: neuronal precursor cells

Treatment protocol

N/A

Growth protocol

Mouse embryonic stem cells (V6.5) were co-cultured with irradiated MEFs (GlobalStem; GSC-6002C) on 0.2% gelatin coated plates in a culture media consisting of Knockout DMEM (Invitrogen; 10829018) containing 10% FBS (GlobalStem; GSM-6002), 1% pen-strep 1% Non-essential amino acids, 1% L-glutamine, 4ul Beta-mercaptoethanol, and .01% LIF (Millipore; ESG1106). mES cells were passaged once on gelatin without MEFs before RNA extraction. V6.5 ES cells were differentiated into neural progenitor cells (NPCs) through embryoid body formation for 4 days and selection in ITSFn media for 5–7 days, and maintained in FGF2 and EGF2 (R&D Systems) as described27. The cells uniformly express nestin and Sox2 and can differentiate into neurons, astrocytes and oligodendrocytes. Mouse lung fibroblasts (ATCC), were grown in DMEM with 10% fetal bovine serum and penicillin/streptomycin at 37°, 5% CO2.

Extracted molecule

total RNA

Extraction protocol

RNA was extracted using the protocol outlined in the RNeasy kit (Qiagen). Extracts were treated with DNase (Ambion 2238). Polyadenylated RNAs was selected using Ambion’s MicroPoly(A)Purist kit (AM1919M) and RNA integrity confirmed using Bioanalyzer (Agilent). A ‘regular’ RNA sequencing library (non strand specific) was created as previously described, with the following modifications.
250 ng of polyA+ RNA was fragmented by heating at 98°C for 33 minutes in 0.2 mM sodium citrate, pH 6.4 (Ambion). Fragmented RNA was mixed with 3 μg random hexamers, incubated at 70°C for 10 minutes, and placed on ice briefly before starting cDNA synthesis. First strand cDNA synthesis was performed using Superscript III (Invitrogen) for 1 hour at 55°C, and second strand using E. coli DNA polymerase and E. coli DNA ligase at 16°C for 2 hours. cDNA was eluted using Qiagen MiniElute kit with 30ul EB buffer. DNA ends were repaired using dNTPs and T4 polymerase, (NEB) followed by purification using the MiniElute kit. Adenine was added to the 3’ end of the DNA fragments to allow adaptor ligation using dATP and Kelnow exonuclease (NEB; M0212S) and purified using MiniElute. Adaptors were ligated and incubated for 15 minutes at room temperature. Phenol/choloform/isoamyl alcohol (Invitrogen 15593-031) extraction followed to remove the DNA ligase. The pellet was then resuspend in 10ul EB Buffer.
The sample was run on a 3% Agarose gel (Nusieve 3:1 Agarose) and a 160 - 380 base pair fragment was cut out and extracted. PCR was performed with Phusion High-Fidelity DNA Polymerase with GC buffer (New England Biolabs) and 2M Betaine (Sigma). [PCR conditions: 30 sec at 98°C, (10 sec at 98°C, 30 sec at 65°C, 30 sec at 72°C -16 cycles) 5 min at 72°C, forever at 4°C], and products were run on a poly-acrylamide gel for 60 minutes at 120 volts. The PCR products were cleaned up with Agencourt AMPure XP magnetic beads (A63880) to completely remove primers and product was submitted for Illumina sequencing.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina Genome Analyzer II

Description

RNASeq of polyA+ RNA from neural progenitor cells, derived from V6.5 embryonic stem cells

Data processing

Reads were aligned against build mm9 using TopHat v1.0.13.