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GEO help: Mouse over screen elements for information. |
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| Status |
Public on May 05, 2016 |
| Title |
iPS_2i_Rep2_RNA_seq_library |
| Sample type |
SRA |
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| Source name |
NPC-derived Induced Pluripotent Stem Cells
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| Organism |
Mus musculus |
| Characteristics |
cell type: NPC-derived Induced Pluripotent Stem Cells
strain: 129SvJae x C57BL/6
genotype/variation: Sox2-eGFP
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| Treatment protocol |
none
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| Growth protocol |
Induced pluripotent stem cells were derived from primary NPCs as described by (Eminli et al., 2008). After reprogramming and initial expansion, iPS cells were cultured on Mitomycin-C inactived MEFs in 2i serum-free media containing LIF, CHIR99021, PD0325901 (Axon Medchem), as previously described (Rais et al., 2013). After 2i expansion, iPS cells were passaged onto 0.1% gelatin to remove contaminating feeder cells.
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| Extracted molecule |
total RNA |
| Extraction protocol |
0.9 million cells were lysed with Trizol (Life Technologies 15596-026) and snap frozen. Total RNA was then extracted and purified using the miRvana miRNA Isolation Kit (Ambion AM 1561). RNA integrity was assessed using the Agilent BioAnalyer and samples with RIN scores >9 were carried forward for sequencing. 350 ng total RNA was prepared for sequencing using the Illumina TruSeq Stranded Total RNA Library Prep kit with RiboZero (Illumina RS-122-2202). Paired-end sequencing was performed on the NextSeq 500 with 75 cycles per end.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NextSeq 500 |
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| Description |
RNA-seq
Beagan_et_al_normalized_10sample_gene_expression_w_coordinates_1_29_2016.txt
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| Data processing |
RNA-seq reads were aligned to the mouse genome (build mm9) using the Tophat (Tophat v2.1.0) alignment tool (Trapnell et al., 2009) with the parameters: -r 100 --no-coverage-search --library-type fr-firststrand and UCSC gene annotations. Gene level read counts were computed using the htseq-count tool (http://www-huber.embl.de/users/anders/HTSeq/doc/count.html) with parameters: -m union --stranded=reverse and UCSC gene annotations.
To generate Beagan_et_al_normalized_10sample_gene_expression_w_coordinates_1_29_2016.txt: In experimental analyses of 10 replicates (ES_Rep1, ES_Rep2, pNPC_Rep1, pNPC_Rep2, iPS_Rep1, iPS_Rep2, ES2i_Rep1, ES2i_Rep2, iPS2i_Rep1, iPS2i_Rep2), genes with more than three counts in at least five libraries were retained, resulting in a total of 11,767 genes analyzed. To account for library-specific differences in sequencing depth, log2-transformed libraries were normalized by read depth of the 75%tile gene. Libraries were assessed for the absence of batch effects before proceeding to downstream biological analyses.
Supplementary_files_format_and_content: RNA-seq processed data files format and content: The processed data file Beagan_et_al_normalized_10sample_gene_expression_w_coordinates_1_29_2016.txt was created for a 10 sample experiment (ES_Rep1, ES_Rep2, pNPC_Rep1, pNPC_Rep2, iPS_Rep1, iPS_Rep2, ES2i_Rep1, ES2i_Rep2, iPS2i_Rep1, iPS2i_Rep2). For each gene, data are listed as log2-transformed, sequencing depth-normalized counts. Prior to downstream analyses, libraries were confirmed to have minimal batch effects.
Supplementary_files_format_and_content: The concatenated counts files (v65ES_Rep1_S11_concatenated_counts_11_29_2015.txt, v65ES_Rep2_S12_concatenated_counts_11_29_2015.txt, v65ESw2i_Rep1_S9_concatenated_counts_11_29_2015.txt, v65ESw2i_Rep2_S10_concatenated_counts_11_29_2015.txt, pNPC_Rep1_S3_concatenated_counts_11_29_2015.txt, pNPC_Rep2_S4_concatenated_counts_11_29_2015.txt, iPS_Rep1_S1_concatenated_counts_11_29_2015.txt, iPS_Rep3_S2_concatenated_counts_11_29_2015.txt, iPSw2i_Rep1_S5_concatenated_counts_11_29_2015.txt, iPSw2i_Rep2_S6_concatenated_counts_11_29_2015.txt) were generated by aligning RNA-seq reads to the mouse genome (build mm9) using the Tophat (Tophat v2.1.0) alignment tool (Trapnell et al., 2009) with the parameters: -r 100 --no-coverage-search --library-type fr-firststrand and UCSC gene annotations. Gene level read counts were computed using the htseq-count tool (http://www-huber.embl.de/users/anders/HTSeq/doc/count.html) with parameters: -m union --stranded=reverse and UCSC gene annotations. After ensuring the absence of batch effects between sequencing runs, counts files for the same sample from two sequencing runs were concatenated to generate the counts files. For each gene, data are listed as mapped counts.
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| Submission date |
Dec 17, 2015 |
| Last update date |
May 15, 2019 |
| Contact name |
Jennifer E Phillips-Cremins |
| E-mail(s) |
jcremins@seas.upenn.edu
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| Organization name |
University of Pennsylvania
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| Department |
Bioengineering
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| Street address |
415 Curie Blvd
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| City |
Philadelphia |
| State/province |
Pennsylvania |
| ZIP/Postal code |
19104 |
| Country |
USA |
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| Platform ID |
GPL19057 |
| Series (1) |
| GSE68582 |
Local Genome Topology Can Exhibit an Incompletely Rewired 3D-Folding State During Somatic Cell Reprogramming |
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| Relations |
| BioSample |
SAMN04348399 |
| SRA |
SRX1488361 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1974114_iPSw2i_Rep2_S6_concatenated_counts_11_29_2015.txt.gz |
179.9 Kb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
| Processed data are available on Series record |
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