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Sample GSM1369183

Query DataSets for GSM1369183

Status

Public on Jul 01, 2014

Title

Day 0 B

Sample type

SRA

Source name

in vitro_hESC_cortecon_day 0

Organism

Homo sapiens

Characteristics

cell type: hESC (WA-09)
Stage: Pluripotency
run id: Cortecon

Treatment protocol

When cultures were at ~90% confluency the culture medium was changed to KSR-medium (all reagents were obtained from Life Technologies unless indicated otherwise): knockout-DMEM with 15% (v/v) KSR, 2mM L-glutamine, 100 µM non-essential amino acids, 55 µM 2-mercaptoethanol, supplemented with 100 nM LDN193189 (Stemgent) and 10 µM SB431542 (Tocris). Medium was changed daily, on the third day 1µM cyclopamine (Stemgent) was added. Medium was changed every other day, as of day five increasing amounts of N2-medium (25%, 50%, 75%, 100%) was added, cyclopamine concentration was maintained at 1 µM. N2-medium (all reagents were obtained from Life Technologies unless indicated otherwise): DMEM/F12 (1:1) with N-2 supplement and 0.775 g glucose (Sigma). In the Test case, the protocol was started when cells were ~65% confluent.

Growth protocol

H9 cells were cultured in HES-medium (all reagents were obtained from Life Technologies): DMEM/F12 (1:1) with 20% (v/v) KSR, 1 mM L-glutamine, 100 µM non-essential amino acids, 55 µM 2-mercaptoethanol, and 10 ng ml-1 FGF-2.

Extracted molecule

total RNA

Extraction protocol

Samples were collected using RNAprotect cell reagent (Qiagen), homogenized with QIAshredder (Qiagen), and total RNA extracted using RNeasy plus mini kit (Qiagen), according to manufacturer’s protocol.
Samples were prepared for RNA-seq (including library preparation, bar-coding, etcetera) by Expression Analysis (Durham, NC, USA), or Covance (Seattle, WA, USA)

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiSeq 2000

Description

processed data file: Cortecon_normalized.csv
214_d0

Data processing

Data was mapped to human genome 19 using BFAST
Mapped files were analyzed utililzing R in conjunction with multiple packages: GenomicFeatures, rtracklayer, Rsamtools, GenomicRanges, edgeR, Mfuzz, DESeq, DESeq2, goseq, SPIA, ggplot2, igraph, RcolorBrewer, GO.db, org.Hs.eg.db, foreach, parallel, multicore, doMC
Genome_build: hg19
Supplementary_files_format_and_content: text, counts normalized for library size with DESeq

Submission date

Apr 15, 2014

Last update date

May 15, 2019

Contact name

Thomas R Kiehl

E-mail(s)

tomkiehl@neuralsci.org

Phone

518-694-8188

Organization name

Neural Stem Cell Institute