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Sample GSM5608871 Query DataSets for GSM5608871
Status Public on Feb 04, 2022
Title H1JA_D2 cNES, biological replicate 1
Sample type RNA
Source name Cortically specified neuroepithelial stem cell clone derived from H1 hESCs
Organism Homo sapiens
Characteristics cell type: cNESCs
Treatment protocol No treatment
Growth protocol H1 and CA1 human pluripotent stem cells (hPSCs) were routinely cultured on mitomycin C treated mouse embryonic fibroblast feeder cells in KSR media with 10ng/ml FGF2. KSR media contains 80% DMEM-F12 (GIBCO), 20% Knockout serum replacement (GIBCO), 0.1mM 2-mercaptoethanol, 2mM Glutamax, 0.1mM Non-essential amino acids. Prior to neural differentiation hPSCs were transferred to geltrex (GIBCO) coated surface in the absence of feeders and were cultured in mTESR2 (Stem cell technologies) media. On the day of neural induction, hPSCs were replated on geltrex coated surface in N2B27 media supplemented with 10µM SB431542 and 100nM LDN193189 and 10µM Y27632. Neural cultures were detached from Geltrex first on day 8-10 with Accutase or PBS-EDTA, and replated on laminin (Sigma) coated surface, in neural maintenance media (NES) supplemented with 10µM Y27632 at 3x105 cells per cm2 and 6F : 10ng/ml FGF2, 3μM CHIR99021, 1μM SB431542 (Peprotech), 100nM LDN193189, 100nM K02288 (Selleckchem), 100nM AKTiVIII, 75nM MK2206 (Selleckchem), and 1 μM XAV939 (Selleckchem). NES media contains 50% DMEM-F12, 50% Neurobasal, 0.1mM 2mercaptoethanol, 2mM Glutamax, 1x N2 supplement, 0.05x B27 minus vitamin A supplement (GIBCO). 4F media is made by supplementing NES media with 10ng/ml FGF2, 3μM CHIR99021, 1μM SB431542 or 50ng/ml FST (Peprotech), 100nM LDN193189 or 50 ng/ml NOGGIN. 6F media contains components of the 4F media with 100nM K02288 (Selleckchem), 100nM AKTiVIII and 75nM MK2206 (Selleckchem), and 1-2 μM XAV939 (Selleckchem). XAV939 concentrations need to be adjusted to each cell line, 1μM for H1, CA1, H9, CTRL hPSCd-erived cNESCs, 2 μM for Shef6 derived cNESCs. For single cell clone isolations, H1 (passage 12) cNES cells were cultured in 4F or 6F medium as described in section ‘Neuroepithelial stem cell maintenance and differentiation’. Three to four days after plating the cells were rinsed with PBS once and detached with 5-minute Accutase digestion at 37°C. The cells were collected with 10fold volume DMEM-F12 with 0.1% BSA and centrifuged at 300xg for 3.5 minutes. The pelleted cells were resuspended in 1 ml fresh 6F media and triturated by pipetting with 1000μl tip for 20 times with moderate force without generating bubbles. Cells were centrifuged with additional 5fold volume DMEM-F12 with 0.1% BSA at 300xg for 3.5 minutes. For single cell cloning cNES cells were plated in 6F media at 100 cells per cm2 culture surface area in a 10 cm dish coated with poly-D-lysine and laminin. FGF was added daily at 20ng/ml concentration without media change for the first 4 days. Media was completely changed after 6 days. Colonies were manually picked at day 10 to 96 wells. CA1 and H1 hESCs were also differentiated to dorsal forebrain neuroepithelium for 8-10d without culture in 6F condition. Mid-hindbrain specified NESCs (mhbNESCs) were derived in FGF and EGF from hPSCs (AF22, 23) or from human embryos (SAI1,3).
Extracted molecule total RNA
Extraction protocol Total RNA was isolated from cultured cells with Qiagen RNeasy kit according to manufacturer’s instructions and was assessed for quality and quantity on a Bio-analyzer
Label biotin
Label protocol Purified RNA was labeled for analysis on Affymetrix Human Transcriptome Array 2.0 (Affymetrix) according to the manufacturer’s instructions, at Microarray Facility of The Centre for Applied Genomics at Sickkids, Toronto, Ontario.
Hybridization protocol Purified and labeled RNA was prepared for analysis on Affymetrix Human Transcriptome Array 2.0 (Affymetrix) according to the manufacturer’s instructions, at Microarray Facility of The Centre for Applied Genomics at Sickkids, Toronto, Ontario.
Scan protocol Prepared RNA was analyzed on Affymetrix Human Transcriptome Array 2.0 (Affymetrix) according to the manufacturer’s instructions, at Microarray Facility of The Centre for Applied Genomics at Sickkids, Toronto, Ontario.
Description H1JA_D2_6
Data processing RMA method normalization, background substraction, and summarization was performed using Oligo (v1.34.2) R package from bioconductor. Normalized gene expression intensities were log2-scaled for all subsequent analyses. Unless otherwise stated, all data presented are representative of at least two independent experiments. A linear model approach and the empirical Bayes statistics were implemented for differential gene expression analysis, as described in limma (v3.26.9) R package user guide. P-values were adjusted using the Benjamini–Hochberg method and significance cut-off was set at 0.01.
Submission date Oct 04, 2021
Last update date Feb 04, 2022
Contact name Samer M.I. Hussein
Organization name CHU de Québec - Université Laval
Department Departement of medecine
Lab Hussein Lab
Street address 9 rue Mcmahon
City Quebec City
State/province Quebec
ZIP/Postal code G1R 3S3
Country Canada
Platform ID GPL17586
Series (1)
GSE185258 Signal requirement for cortical potential of transplantable human neuroepithelial stem cells [array]

Data table header descriptions
VALUE RMA-normalized signal intensity

Data table
2824546_st 12.55828087
2824549_st 11.85209235
2824551_st 12.46691035
2824554_st 12.19642034
2827992_st 11.48783475
2827995_st 11.43579357
2827996_st 11.56529183
2828010_st 10.13024869
2828012_st 9.81588217
2835442_st 10.9682968
2835447_st 7.993091908
2835453_st 9.855672442
2835456_st 12.50248893
2835459_st 11.59033421
2835461_st 10.27642533
2839509_st 12.73441619
2839511_st 12.01126019
2839513_st 11.83231177
2839515_st 10.80579077
2839517_st 11.80456408

Total number of rows: 70523

Table truncated, full table size 1930 Kbytes.

Supplementary file Size Download File type/resource
GSM5608871_HTA2_030716H_AN5_6.CEL.gz 25.6 Mb (ftp)(http) CEL
Processed data included within Sample table

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