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Sample GSM1974106 Query DataSets for GSM1974106
Status Public on May 05, 2016
Title v6.5_Rep2_RNA_seq_library
Sample type SRA
 
Source name V6.5 ES cells
Organism Mus musculus
Characteristics cell type: V6.5 ES cells
strain: 129SvJae x C57BL/6
gender: male
Treatment protocol none
Growth protocol Murine V6.5 ES cells were expanded on Mitomycin-C-inactivated MEF feeder layers in pluripotent media supplemented with LIF. After initial expansion, ES cells were passaged onto 0.1% gelatin to remove contaminating feeder cells.
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.
 
Library strategy RNA-Seq
Library source transcriptomic
Library selection cDNA
Instrument model Illumina NextSeq 500
 
Description RNA-seq
Beagan_et_al_normalized_10sample_gene_expression_w_coordinates_1_29_2016.txt
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.
 
Submission date Dec 17, 2015
Last update date May 15, 2019
Contact name Jennifer E Phillips-Cremins
E-mail(s) jcremins@seas.upenn.edu
Organization name University of Pennsylvania
Department Bioengineering
Street address 415 Curie Blvd
City Philadelphia
State/province Pennsylvania
ZIP/Postal code 19104
Country USA
 
Platform ID GPL19057
Series (1)
GSE68582 Local Genome Topology Can Exhibit an Incompletely Rewired 3D-Folding State During Somatic Cell Reprogramming
Relations
BioSample SAMN04348391
SRA SRX1488353

Supplementary file Size Download File type/resource
GSM1974106_v65ES_Rep2_S12_concatenated_counts_11_29_2015.txt.gz 172.8 Kb (ftp)(http) TXT
SRA Run SelectorHelp
Raw data are available in SRA
Processed data provided as supplementary file
Processed data are available on Series record

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