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| Status |
Public on Feb 21, 2024 |
| Title |
E15.5_Mut_8627_F [Mutant_8627_F] |
| Sample type |
SRA |
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| Source name |
E15.5 whole pancreas
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| Organism |
Mus musculus |
| Characteristics |
tissue: E15.5 whole pancreas
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| Treatment protocol |
Selected samples were treated with doxycycline (DOX) for 14-16hrs prior to collection. See SAMPLES section.
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| Growth protocol |
Inducible ESC lines were cultured and generated as previously described (Mazzoni et al., 2011). Inducible lines created in this study include wildtype NKX2.2 (iNKX2.2) and SD-mutant NKX2.2 (iSDmut), which harbors the same mutations as the Nkx2.2SDmut mice described above. iNKX2.2 and iSDmut ESC lines were differentiated into MNs as previously described (Wichterle et al., 2002). The first day of the differentiation protocol was referred to as Day 0. To induce expression of the transgenes in OLIG2+ pMN, DOX (1µg/ml) was added on late Day 3 for ~16hrs.
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| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was extracted with the RNeasy Mini Kit (Qiagen, 74104).
RNA libraries were prepared for sequencing using Illumina Trueseq RNA Prep Kit.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NovaSeq 6000 |
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| Description |
E15.5_pancreas_RNAseq_readcounts.txt
E15.5_pancreas_RNAseq_SigGenes.txt
E15.5_pancreas_RNAseq_Normalized_readcounts.txt
Mutant_8627_F
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| Data processing |
RNA-seq files
Reads were mapped to the mouse genome (mm10) using the RNA-seq alignment algorithm in the STAR (Spliced Transcripts Alignment to a Reference) software package (version 2.3.1) (Dobin et al., 2013).
To estimate the relative abundances of transcripts, Cufflinks software was used (version 2.2.1) (Trapnell et al., 2013). FPKM values (Fragments Per Kilobase of exon per Million fragments mapped) were calculated per individual gene.
Differential RNA expression across cohorts was assessed using DESeq2 (Love et al., 2014) for E15.5 samples. All other samples were analyzed using EdgeR (version 3.20.9) and limma (version 3.34.9) software (Phipson et al., 2016; Ritchie et al., 2015; Robinson et al., 2010).
Assembly: mm10
Supplementary files format and content: raw readcount text file used as input into DESeq2/EdgeR/limma
Supplementary files format and content: text files of results of DESeq2/EdgeR/limma analysis
Supplementary files format and content: text file of abundance measurements (FPKM values)
ChIP-seq files
Peak calling of Nkx2.2 ChIP-seq carried out using the GEM algorithm (Guo et al., 2012).
Read-count intensity was obtained within +/- 500bp of all GEM identified peaks. Read counts of individual replicates were then normalized to their respective sequencing library size, and only loci above a given threshold in both replicates were used for subsequent analysis.
GEM peaks were then further processed as follows. If multiple peaks resided within 500bp of each other, they were merged into one peak using Bedtools (version 2.17.0) (Quinlan, 2010). The locus with the highest read density (+/- 100bp) was identified using Tidyverse (version 1.2.1) (Wickham H., 2019) and utilized as the center of the occupied region. Read counts for replicates were then obtained within +/- 500bp from the center of these merged peaks. The new 500bp readcount dataset was then imported into the EdgeR (version 3.20.9) and limma (version 3.34.9) software packages (Phipson et al., 2016; Ritchie et al., 2015; Robinson et al., 2010).
Final readcounts for all peaks were converted to counts per million (CPM), quantile-normalized, log2 transformed (logCPM), and then averaged. The average logCPM value for each peak was used as a measurement of the level of Nkx2.2 occupancy in that region.
Supplementary files format and content: peak text files generated by GEM software
Supplementary files format and content: raw readcount text files for peaks used as input into EdgeR/limma software and to make logCPM files
Supplementary files format and content: logCPM text files for all peaks used in EdgeR/limma analysis
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| Submission date |
Feb 28, 2023 |
| Last update date |
Feb 21, 2024 |
| Contact name |
Elena Abarinov |
| E-mail(s) |
elenavabarinov@gmail.com
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| Organization name |
University of Colorado at Denver
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| Street address |
1775 Aurora Ct
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| City |
Aurora |
| State/province |
Colorado |
| ZIP/Postal code |
80045 |
| Country |
USA |
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| Platform ID |
GPL24247 |
| Series (1) |
| GSE226345 |
Global profiling of Nkx2.2 SD-domain mutant pancreas during development and adulthood compared to transcriptional profiling of SD-domain mutant expressing neural progenitors. |
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| Relations |
| BioSample |
SAMN33546512 |
| SRA |
SRX19531372 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
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