cell type: lymphoblast cell line disease state: Schizophrenia and epilepsy id (rutgers): 05C48584 nimh identifier: 143-2330-001 sex: M
Growth protocol
Lymphoblastoid cell lines were establihsed and maintained by Rutgers University for the NIMH
Extracted molecule
genomic DNA
Extraction protocol
DNA was isolated from lymphoblastoid cell lines by Rutgers University by desalting using the Autopure LS robotic workstation with Autopure reagents (Qiagen, Inc., Valencia, CA)
Label
Cy5
Label protocol
The digested DNA was labeled with Bioprime Array CGH Genomic Labeling System following the manufacturer’s recommended protocol with minor additions (Invitrogen, Carlsbad, CA). Following the addition of random primers to the digested DNA, the mixture was boiled for 5 minutes then cooled on ice for 5 minutes. Cyanine 5-dCTP (for the experimental sample), cyanine 3-dCTP (for the reference sample) (PerkinElmer, Boston, MA), dCTP nucleotide mix, and exo-Klenow fragment (40 units/µL) were added and the reaction was incubated at 37°C for 2 hours then incubated at 65°C for10 minutes to inactivate the enzyme. The labeled product was purified from unincorporated nucleotides using Millipore columns or plate (Millipore, Billerica, MA, USA)
sex: M sample type: reference-1 tissue: blood disease state: control
Growth protocol
Lymphoblastoid cell lines were establihsed and maintained by Rutgers University for the NIMH
Extracted molecule
genomic DNA
Extraction protocol
DNA was isolated from lymphoblastoid cell lines by Rutgers University by desalting using the Autopure LS robotic workstation with Autopure reagents (Qiagen, Inc., Valencia, CA)
Label
Cy3
Label protocol
The digested DNA was labeled with Bioprime Array CGH Genomic Labeling System following the manufacturer’s recommended protocol with minor additions (Invitrogen, Carlsbad, CA). Following the addition of random primers to the digested DNA, the mixture was boiled for 5 minutes then cooled on ice for 5 minutes. Cyanine 5-dCTP (for the experimental sample), cyanine 3-dCTP (for the reference sample) (PerkinElmer, Boston, MA), dCTP nucleotide mix, and exo-Klenow fragment (40 units/µL) were added and the reaction was incubated at 37°C for 2 hours then incubated at 65°C for10 minutes to inactivate the enzyme. The labeled product was purified from unincorporated nucleotides using Millipore columns or plate (Millipore, Billerica, MA, USA)
Hybridization protocol
The nanodrop fluorospectrometer was used to measure DNA concentrantion and Dye incorporation inorder to pair subjects with controls according to the following criteria: pmoles of dye were required to be within 3 units and DNA concentration within 100ng/uL. For each hybridization, the labeled experimental and reference DNAs were boiled for 3 minuted and incubated at 37°C for 30-90 minutes with human Cot-1 DNA (Invitrogen, Carlsbad, CA), blocking agent and hybridization buffer (Agilent Technologies, Santa Clara, CA). The samples were then added to an array, which was placed inside a microarray hybridization chamber (Agilent Technologies, Santa Clara, CA), and allowed to hybridize at 65°C for 35-68 hours in a rotating hybridization oven (Agilent Technologies, Santa Clara, CA). Following hybridization, slides were washed following the manufacturer’s protocol (Agilent Technologies).
Scan protocol
Agilent G2565 DNA Microarray Scanner (Agilent Technologies, Santa Clara, CA) with 3 um resolution.
Description
n/a
Data processing
scanned images were quantified using the Agilent Feature Extraction (FE) software (version 10.7.3.1, Agilent Technologies)
