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| Status |
Public on May 05, 2016 |
| Title |
iPS_2i_Rep1_5C_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. Cells were grown to ~7e6 cells per 15 cm dish at the time of fixation with 1% formaldehyde before downstream assay.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
3C templates were generated using HindIII as previously described (Dekker et al., 2002; van Berkum and Dekker, 2009). 5C libraries were generated from 3C templates using an alternating primer design across 1-2 Mb regions around Oct4, Nanog, Sox2, Nestin, Olig1-Olig2, Klf4, and a gene desert negative control (described previously Dostie and Dekker, 2007; Dostie et al., 2006; van Berkum and Dekker, 2009).
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| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina NextSeq 500 |
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| Description |
Chromosome-Conformation-Capture-Carbon-Copy (5C) Library
BED_ES-NPC-iPS-LOCI_mm9.bed
Beagan_et_al_5C_processed_data_file_2_23_2016_10samples.txt
BED_binned_mm9_20kbbin_4kbstep-10samples.bed
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| Data processing |
Paired-end reads were aligned to a pseudo-genome consisting of all 5C primers using Bowtie (http://bowtie-bio.sourceforge.net/index.shtml) (Langmead, 2010). Only reads with one unique alignment were considered for downstream analyses. Interactions were counted when both paired-end reads could be uniquely mapped to the 5C primer pseudo-genome. Only interactions between forward-reverse primer pairs were tallied as a true count. Counts were converted to contact matrices for each genomic region queried (i.e. ~1-2 Megabase regions around developmentally regulated genes Nanog, Oct4, Sox2, Nestin, Olig, Klf4) to generate the raw .counts file for each sample.
To correct for bias related to intrinsic properties of restriction fragments (i.e. G-C content; fragment size) and procedural batch effects, we converted our raw .counts data to ‘Observed’ values through a series of pre-processing steps. Briefly, raw .counts matrices were (i) trimmed of primers with less than 100 total counts in any replicate, (ii) quantile normalized across biological conditions, (iii) normalized for primer biases as previously described (Phillips-Cremins et al. 2013), (iv) trimmed of low information primer-primer pairs as described below, (v) log transformed, (vi) binned into 4 kilobase(kb)-sized bins and (vii) smoothed using a sliding 20 kb smoothing windows with 4 kb step-size. A primer-primer pair was set to NaN and removed from downstream analyses if it did not cross a threshold of 10 counts in any one library. Four kb bins were withheld from downstream processing if greater than 80% of the primer-primer pairs within that bin’s smoothing window were NaN. Next, we developed an empirical ‘Expected’ model of the distance-dependent background level of non-specific chromatin interactions. We computed our ‘Expected’ values locally (i.e. for each developmentally regulated region independently) by averaging the observed values across all bin-bin pairs representing equidistant interactions in each region. We then assigned each bin-bin pair a “log2(Observed/Expected)” value by subtracting the region-specific Expected value for a given bin-bin contact distance from the Observed value of bins at the same distance. To compute p-values for each individual bin-bin pair, we modeled our ‘Observed over Expected’ values as a Logistic distribution with location/scale parameters computed independently for each region and each biological replicate. The resulting ‘Interaction Score’ (computed as -10*log2(p-value)) was comparable within and between replicates and allowed for robust detection of fragment-to-fragment looping interactions that were significant above the expected background signal for each genomic region. The processed data files Beagan_et_al_5C_processed_data_file_2_23_2016_6samples.txt and Beagan_et_al_5C_processed_data_file_2_23_2016_10samples.txt were created for 6 sample (V6.5_Rep1, V6.5_Rep2, pNPC_Rep1, pNPC_Rep2, iPS_Rep1, iPS_Rep2) and 10 sample (V6.5_Rep1, V6.5_Rep2, pNPC_Rep1, pNPC_Rep2, iPS_Rep1, iPS_Rep2, V6.52i_Rep1, V6.52i_Rep2, iPS2i_Rep1, iPS2i_Rep2) experimental analyses.
Supplementary_files_format_and_content: The processed data files (Beagan_et_al_5C_processed_data_file_2_23_2016_6samples.txt, Beagan_et_al_5C_processed_data_file_2_23_2016_10samples.txt) contain the following information for each bin-bin pair: “Chromosome” (chromosome containing the 5C region), “Region” (5C Region), “Bin1 ID” (unique identifier of the downstream bin), “Bin2 ID” (unique identifier of the upstream bin), “Bin1 Start” (genomic coordinate of the start of the downstream bin), “Bin1 End” (genomic coordinate of the end of the downstream bin), “Bin2 Start” (genomic coordinate of the start of the upstream bin), “Bin2 End” (genomic coordinate of the end of the upstream bin), “Distance” (mid-to-mid distance between interaction bins), “_obs.counts” (normalized, logged, binned and smoothed interaction counts between Bin1 and Bin2), “_exp.counts” (expected interaction counts as determined by distance-dependent expected model), “_obs_over_exp.counts” (calculated by subtracting Expected counts from Observed counts) “_obs_over_exp_pvalues.counts” (p-value for a specific bin-bin interaction calculated by fitting Observed over Expected counts to a Logistic distribution). The processed data files Beagan_et_al_5C_processed_data_file_2_23_2016_6samples.txt and Beagan_et_al_5C_processed_data_file_2_23_2016_10samples.txt were created for 6 sample (V6.5_Rep1, V6.5_Rep2, pNPC_Rep1, pNPC_Rep2, iPS_Rep1, iPS_Rep2) and 10 sample (V6.5_Rep1, V6.5_Rep2, pNPC_Rep1, pNPC_Rep2, iPS_Rep1, iPS_Rep2, V6.52i_Rep1, V6.52i_Rep2, iPS2i_Rep1, iPS2i_Rep2) experimental analyses. NOTE: Figures comparing chromatin architecture between only ES, NPC, and iPS cells were generated using the ‘6 sample’ processed data, while figures including the ES in 2i and iPS in 2i conditions were generated from the ’10 sample’ processed data.
Supplementary_files_format_and_content: The raw primer bed file (BED_ES-NPC-iPS-LOCI_mm9.bed) contains the following information for each individual primer used in the 5C experiment: “Chromosome” (chromosome containing the 5C region), “Start Site” (genomic coordinate of the start of the primer fragment), “End Site” (genomic coordinate of the end of the primer fragment), “Primer ID” (unique identifier within our primer set).
Supplementary_files_format_and_content: The binned primer bed file (BED_binned_mm9_20kbbin_4kbstep-6samples.bed, BED_binned_mm9_20kbbin_4kbstep-10samples.bed) contains the following information for each bin: “Chromosome” (chromosome containing the 5C region), “Start Site” (genomic coordinate of the start of the bin), “End Site” (genomic coordinate of the end of the bin), “Bin ID” (unique identifier within our primer set). BED_binned_mm9_20kbbin_4kbstep-6samples.bed and BED_binned_mm9_20kbbin_4kbstep-10samples.bed were created for 6 sample (V6.5_Rep1, V6.5_Rep2, pNPC_Rep1, pNPC_Rep2, iPS_Rep1, iPS_Rep2) and 10 sample (V6.5_Rep1, V6.5_Rep2, pNPC_Rep1, pNPC_Rep2, iPS_Rep1, iPS_Rep2, V6.52i_Rep1, V6.52i_Rep2, iPS2i_Rep1, iPS2i_Rep2) experimental analyses.
Supplementary_files_format_and_content: The raw counts files (v65_Rep1.counts, v65_Rep2.counts, v65_2i_Rep1.counts, v65_2i_Rep2.counts, pNPC_Rep1.counts, pNPC_Rep2.counts, iPS_Rep1.counts, iPS_Rep2.counts, iPS_2i_Rep1.counts, iPS_2i_Rep2.counts) were generated by first aligning paired-end reads to a pseudo-genome consisting of all 5C primers using Bowtie (http://bowtie-bio.sourceforge.net/index.shtml) (Langmead, 2010). Only reads with one unique alignment were considered for downstream analyses. Interactions were counted when both paired-end reads could be uniquely mapped to the 5C primer pseudo-genome. Only interactions between forward-reverse primer pairs were tallied as a true count. Counts were converted to contact matrices for each genomic region queried (i.e. ~1-2 Megabase regions around developmentally regulated genes Nanog, Oct4, Sox2, Nestin, Olig, Klf4). The raw counts files (v65_Rep1.counts, v65_Rep2.counts, v65_2i_Rep1.counts, v65_2i_Rep2.counts, pNPC_Rep1.counts, pNPC_Rep2.counts, iPS_Rep1.counts, iPS_Rep2.counts, iPS_2i_Rep1.counts, iPS_2i_Rep2.counts) contain the following information for each primer-primer pair used for downstream analyses: “Forward primer ID” (the forward primer in this primer-primer pair), “Reverse primer ID” (the reverse primer in this primer-primer pair), “Count” (the number of mapped reads to this primer-primer pair).
Supplementary_files_format_and_content: The final interaction classification file (Beagan_et_al_5C_processed_classification_data_file_2_23_2016_6samples.txt) contains the following information for each bin-bin pair that falls into one of our classifications: “Chromosome” (chromosome containing the 5C region), “Region” (5C Region), “Classification”
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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 |
SAMN04348387 |
| SRA |
SRX1488350 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1974103_ips_2i_Rep1.counts.txt.gz |
2.2 Mb |
(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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