cell type: neuron progenitor cells (NPC) disease group: healthy control
Treatment protocol
Procedures are described in Paulsen et al. (2011). For generation of iPS cells, human primary fibroblasts were transduced overnight with equal amounts of concentrated supernatants containing each of the four retroviruses, supplemented with 8 μg/ml polybrene. Twenty-four hours after transduction, virus-containing medium was replaced by the second round of supernatant. Two days after transduction, 50 μg/ml of L-ascorbic acid (USB Corporation) was added to cultures. Six days after transduction, cells were replated (5 × 10^4 cells per six-well plate) on mitotically inactivated mouse embryonic fibroblasts (MEFs). In the following day, medium was replaced by mTeSRTM1 and changed every day. Eight days after transduction, 1 mM of valproic acid (VPA, Sigma) was added to the medium for another 7 days. About 20 days after transduction, colonies were picked up and transferred to six-well plates coated with Matrigel in mTeSRTM1. Pluripotent-like colonies were selected based on morphological criteria, and pluripotency was verified by RT-PCR analyses and immunostaining assays using OCT3/4 (Santa Cruz Biotechnology; mouse, 1:100), stage-specific embryonic antigen 4 (SSEA4; Chemicon; mouse, 1:100), TRA-1-60 (Chemicon; mouse, 1:100), and TRA-1-81 (Chemicon; mouse, 1:100) primary antibodies. Generation of neural precursor cells (NPCs) was performed using retinoic acid (RA), and immunological analyses were done using primary antibodies for nestin (Chemicon; mouse, 1:100), βIII-tubulin (Sigma; mouse, 1:100), and NeuN (Chemicon; mouse, 1:100). The cell lines derived in this work, as well as controls, are available in the cell bank of the National Laboratory for Embryonic Stem Cell Research at Federal University of Rio de Janeiro, LaNCE-RJ (Brazil).
Growth protocol
NA
Extracted molecule
total RNA
Extraction protocol
Total RNA was isolated using TRIzol (Sigma) according to the manufacturer’s instructions. RNA quantity and integrity were assessed by spectrophotometry (Nanodrop) and microfluidics-based electrophoresis (Bioanalyzer, Agilent 2100), respectively, and obtained OD of ~2.0 and RIN>9.
Label
Cy3
Label protocol
Samples were prepared according to the Agilent mRNA Microarray System protocol using 250ng of total RNA for amplification and labelling using the Agilent Low RNA Input Fluorescent Linear Amplification Kit.
cell type: human induced pluripotent stem cells (hiPSC) disease group: healthy control
Treatment protocol
Procedures are described in Paulsen et al. (2011). For generation of iPS cells, human primary fibroblasts were transduced overnight with equal amounts of concentrated supernatants containing each of the four retroviruses, supplemented with 8 μg/ml polybrene. Twenty-four hours after transduction, virus-containing medium was replaced by the second round of supernatant. Two days after transduction, 50 μg/ml of L-ascorbic acid (USB Corporation) was added to cultures. Six days after transduction, cells were replated (5 × 10^4 cells per six-well plate) on mitotically inactivated mouse embryonic fibroblasts (MEFs). In the following day, medium was replaced by mTeSRTM1 and changed every day. Eight days after transduction, 1 mM of valproic acid (VPA, Sigma) was added to the medium for another 7 days. About 20 days after transduction, colonies were picked up and transferred to six-well plates coated with Matrigel in mTeSRTM1. Pluripotent-like colonies were selected based on morphological criteria, and pluripotency was verified by RT-PCR analyses and immunostaining assays using OCT3/4 (Santa Cruz Biotechnology; mouse, 1:100), stage-specific embryonic antigen 4 (SSEA4; Chemicon; mouse, 1:100), TRA-1-60 (Chemicon; mouse, 1:100), and TRA-1-81 (Chemicon; mouse, 1:100) primary antibodies. Generation of neural precursor cells (NPCs) was performed using retinoic acid (RA), and immunological analyses were done using primary antibodies for nestin (Chemicon; mouse, 1:100), βIII-tubulin (Sigma; mouse, 1:100), and NeuN (Chemicon; mouse, 1:100). The cell lines derived in this work, as well as controls, are available in the cell bank of the National Laboratory for Embryonic Stem Cell Research at Federal University of Rio de Janeiro, LaNCE-RJ (Brazil).
Growth protocol
NA
Extracted molecule
total RNA
Extraction protocol
Total RNA was isolated using TRIzol (Sigma) according to the manufacturer’s instructions. RNA quantity and integrity were assessed by spectrophotometry (Nanodrop) and microfluidics-based electrophoresis (Bioanalyzer, Agilent 2100), respectively, and obtained OD of ~2.0 and RIN>9.
Label
Cy5
Label protocol
Samples were prepared according to the Agilent mRNA Microarray System protocol using 250ng of total RNA for amplification and labelling using the Agilent Low RNA Input Fluorescent Linear Amplification Kit.
Hybridization protocol
Samples were hybridized for microarrays in the Agilent SureHyb chambers (Agilent Technologies) for 20 h at 55°C, and the platforms were washed with the manufacturer’s washing buffers.
Scan protocol
Scanned images of the arrays were processed using Feature Extraction 10.7.3.1 software (Agilent Technologies) with default parameters.
Description
Technical replicate of healthy control.
Data processing
Self-self experiments were performed by labelling the SCZP hiPSC with either Cy3 or Cy5 dyes and hybridizing simultaneously on the same microarray slide to determine the intensity-dependent cut-offs. These cut-offs were applied to the non-self-self experiments, (NPCxhIPSC SCZP) and (NPCxhIPSC CON). R (programming language) and Bioconductor package Limma were used.
