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| Status |
Public on Aug 03, 2015 |
| Title |
Undiff_2 |
| Sample type |
SRA |
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| Source name |
CRXp-GFP H9 hES cells
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| Organism |
Homo sapiens |
| Characteristics |
cell type: hES cells
day post induction: Day 0
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| Growth protocol |
H9 hESCs were passaged every 4-5 days at 1:6 ratio by incubating with 2 mg/ml dispase (Invitrogen) at 37°C for 10 min. Colonies were washed, broken into small clumps of cells by pipetting and cultured at 37°C, 5% CO2.
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| Extracted molecule |
total RNA |
| Extraction protocol |
The differentiation was performed according previous report with some modification (Nakano, Cell Stem Cell 2012). Human ESCs were dissociated by Accumax™ (Stem cell technologies) containing 10 µM Y-27632. Basal differentiation medium contained GMEM, 20 % (v/v) KSR, 0.1 mM nonessential amino acids, 1mM pyruvate 100 U/ml penicillin and 100 mg/ml streptomycin (Sigma-Aldrich). Cells on day 0 were quickly aggregated in low attachment 96 well-plates with V-bottom shape (NOF corporation) (9,000 cells/well in 100 µl) containing basal differentiation medium plus 3 µM IWR-1-endo (Wnt inhibitor, Calbiochem) and 20 µM Y-27632. Ten microliter of 1.8 µg/µl of Matrigel (growth factor reduced, BD Biosciences) solution was added in day-2 cultures. Aggregates were maintained for further 4 days when half medium was changed with medium containing 3 µM IWR-1-endo and 180 µg/ml and cultured for 6 days. Day-12 aggregates were transferred to low attachment 90 mm Dishes (NOF corporation) containing basal differentiation medium, 10% (v/v) FBS (Atlanta) and 100 nM Smoothened Agonist (SAG) (hedgehog agonist, Enzo Life Sciences) and maintained for 6 days. On day 18 medium was switched to maintenance medium which was DMEM/F12 containing 1% N2 supplement (Invitrogen), 10% (v/v) FBS, 0.5 µM retinoic acid (Sigma-Aldrich), 0.25 mg/ml Fungizone (GIBCO), 100 U/ml penicillin, and 100 mg/ml streptomycin. Human ESC-NR were grown in maintenance medium in bacterial-grade Dishes from day 18 at 40% O2 and 5% CO2 with medium changed every three day until sample collection.
Total RNA was extracted by RNA purification kit (Norgen Biotek).
Libraries were constructed using a stranded modification of the Illumina TruSeq mRNA (Brooks, et al. Meth Mol Biol 2012). Each library was single-end sequenced in an independent lane of a GAIIx at a length of 76 bases. Fastq files were generated from reads passing chastity filter.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina Genome Analyzer IIx |
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| Description |
undifferentiated CRXp-GFP HP hES cells
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| Data processing |
Basecalls and quality scores performed using RTA 1.13.48 and Fastq files generated from reads passing chastity filtering.
The reference assembly GRCh37.p13 with transcript annotation Gencode 19 was used for alignment and quantitation. Transcript quantitation was accomplished by streaming read alignment from Bowtie2 (Langmead and Salzberg, 2012) to eXpress (Roberts and Pachter, 2013; Roberts et al., 2013). Bowtie2 alignment settings were used as previously described (Roberts et al., 2013) and eXpress setting –r-stranded was used for directional transcript analysis. Bias corrected effective counts were used for downstream normalization and differential expression analysis. Read alignments used for viewing in Integrated Genomics Viewer (IGV) (Thorvaldsdottir et al., 2013) were performed using a 2-step alignment strategy employing TopHat2 (Kim et al., 2013) splice junction mapper keeping the alignment with the best alignment score. Reads were first aligned to the annotation GTF prior to aligning to the genome reference assembly using the default settings with the exception of the following options: -g 1 –library-type fr-firststrand –G <gtf file>.
Effective counts from eXpress were used to filter and to normalize the data via library size correction using the TMM method within the R package edgeR. Prior to normalization, transcripts showing at least 1 Count Per Million (CPM) in at least half of the samples within each time point were kept for further analysis. Design matrices were created for defining groups (time course) with and without batch definition, however, only the time course factor was used for TMM normalization. Limma “RemoveBatchEffect” function was executed on log2 transformed CPM data. Batch corrected log2 CPM values were then converted to FPKM values prior to clustering. Affinity Propagation (AP) clustering was used to assess the success of the procedure. Row-wise average of undifferentiated sample values was added as the first column to the processed data after differential expression analysis.
Differential expression was performed using the edgeR likelihood ratio test from on a generalized linear model (glm), with day 37 as the intercept. In the glm, the batch factor was used as a blocking factor. Significance was defined as having a false discovery rate (FDR) less than or equal to 0.05, and 4697 transcripts fit this criterion. Volcano plots of those comparisons were plotted to see the ideal fold change cutoff. The colors of the points those represent the mean expression of the gene between the time points compared. The key observation is that the mean expression of a transcript is getting closer to 0 (blue) as the fold change is getting closer to 0 although all the FDRs in the plot equal 0.05. We decided to restrict our interest to the transcripts having greater than a 2-fold change, therefor, 3241 transcripts were left after this filtering. The boxplot of the remaining transcripts on batch corrected RPKM values was still showing the median expression around 4 RPKM. We applied another filter eliminating the transcripts where their maximum expression values were less than the median of the medians of the remaining transcripts. The final number of the transcripts used for further analysis was 3157.
Genome_build: GRCh37.p13 with Gencode 19
Supplementary_files_format_and_content: GFPneg_eXpress.counts.csv: GFP negative effective counts output from eXpress.
Supplementary_files_format_and_content: GFPpos_eXpress.counts.csv: GFP positive effective counts output from eXpress.
Supplementary_files_format_and_content: Normalized_FPKM.xls: Batch corrected and normalized FPKM values (log2(FPKM+1).
Supplementary_files_format_and_content: DE_transcripts.xls: Differentially expressed transcript list with averaged FPKM values (log2(FPKM+1).
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| Submission date |
Apr 07, 2015 |
| Last update date |
May 15, 2019 |
| Contact name |
Matthew J Brooks |
| E-mail(s) |
brooksma@nei.nih.gov
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| Phone |
301-443-4906
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| Organization name |
NIH
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| Department |
NEI
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| Lab |
NNRL
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| Street address |
6 Center Dr Bldg 6, Rm 303
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| City |
Bethesda |
| State/province |
MD |
| ZIP/Postal code |
20892 |
| Country |
USA |
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| Platform ID |
GPL10999 |
| Series (1) |
| GSE67645 |
Transcriptome dynamics of developing photoreceptors in 3-D retina cultures recapitulates temporal sequence of human cone and rod differentiation revealing cell surface markers and gene networks |
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| Relations |
| BioSample |
SAMN03465113 |
| SRA |
SRX982257 |
| Supplementary data files not provided |
SRA Run Selector |
| Processed data are available on Series record |
| Raw data are available in SRA |
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