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| Status |
Public on May 22, 2017 |
| Title |
Control_knockdown_Rep2_5C_library |
| Sample type |
SRA |
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| Source name |
Neural Progenitor Cells
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| Organism |
Mus musculus |
| Characteristics |
cell type: Neural Progenitor Cells
developmental stage: P1
strain: 129SvJae x C57BL/6; Sox2-eGFP
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| Growth protocol |
Neural progenitor cells were isolated from whole brains of P1 129SvJae x C57/BL6, Sox2-eGFP mice and cultured as neurospheres in Neural Stem Cell media: DMEM/F12 media (Invitrogen 12100-046 and 21700-075) containing 72 mM glucose, 120 mM Sodium Bicarbonate, 5.6 mM Hepes (Sigma H-0887), 27.5 nM Sodium Selenite (Sigma S-9133), 18 nM progesterone (Sigma P0130), 90 ug/mL Apo-transferrin (Sigma T1428), 23 ug/mL insulin (Sigma I6634), 100 uM putrescine (Sigma P-7505), 2 mM L-glutamine (Gibco 25030-081), 1% Pen/Strep (Sigma P0781), 2 ug/mL heparin, 20 ng/mL rhEGF (R&D Systems) , and 10 ng/mL rhFGF (R&D systems). Neurospheres were passaged every 3-4 days to prevent the formation of necrotic cores. After two passages, neurospheres were dissociated with Accutase and plated on Poly-D-Lysine Hydrobromide (100 ug/mL, Sigma P6407), and laminin (8 ug/mL, Corning 354232) coated plates at 20,000 cells/cm^2. After allowing the cells to adhere and grow, cells were treated with 20 nM siRNA targeting YY1 (Dharmacon # L-050273-00-0005) for 78 hours, replacing the media and siRNA every 24 hours. Cells were fixed for 3C with formaldehyde at the conclusion of 78 hours.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
3C templates were generated using HindIII with an in situ 3C protocol similar to that previously described in Rao et al. 2014. 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
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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). Only the Sox2 and Klf4 regions were queried in the YY1 and Control siRNA samples.
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 10 total counts in any replicate, (ii) quantile normalized across biological conditions, (iii) normalized for primer biases using an adaptation of the 'Express' matrix-balancing algorithm (Sauria 2015) , (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 16 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 at least three replicates. 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 local background level of non-specific chromatin interactions. We computed our ‘Expected’ values locally by first averaging the observed values across all bin-bin pairs representing equidistant interactions, then for each bin-bin pair correcting this distance-dependence expected with local 'Donut' and 'Lower Left' correction factors as described previously (Rao 2014); we retained the maximum of the Donut and Lower Left background calculations as our 'Expected' value for that bin-bin pair. 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 file Beagan_et_al_Processed_Data_File_2_22_2017.txt was created for 6 sample (ES 2i Rep1, ES 2i Rep2, ES Serum Rep1, ES Serum Rep2, pNPC Rep1, pNPC Rep2) experimental analyses. The processed data file Beagan_et_al_YY1_Knockdown_5C_Processed_Data_File_2_22_2017.txt was created for 4 sample (Control_siRNA_Rep1, Control_siRNA_Rep2, YY1_siRNA_Rep1, YY1_siRNA_Rep2) experimental analyses.
Genome_build: mm9
Supplementary_files_format_and_content: The processed data files Beagan_et_al_Processed_Data_File_2_22_2017.txt and Beagan_et_al_YY1_Knockdown_5C_Processed_Data_File_2_22_2017.txt contain the following information for each bin-bin pair: “Region” (5C Region), “Bin1 ID” (unique identifier of the downstream bin), “Bin2 ID” (unique identifier of the upstream bin), “Chromosome” (chromosome containing the 5C region), “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), “_observed.counts” (normalized, logged, binned and smoothed interaction counts between Bin1 and Bin2), “_expected.counts” (expected interaction counts as determined by the maximum of the Donut and Lower Left local background models), “_obs_over_max_donut_ll.counts” (calculated by subtracting Expected counts from Observed counts) “_obs_over_max_donut_ll_pvalues.counts” (p-value for a specific bin-bin interaction calculated by fitting Observed over Expected counts to a Logistic distribution), "_obs_over_exp_int_scores_diag_trimmed.counts" (-10*log2(p-value) of each p-value, with on diagonal pixels trimmed - used to plot interaction score heatmaps). The processed data files Beagan_et_al_Processed_Data_File_2_22_2017.txt also contains an additional column "obs_over_exp_int_scores_diag_cluster_trimmed.counts" from which noisey loops have been removed - this file is used to calculate annotation enrichments, etc.The processed data file Beagan_et_al_Processed_Data_File_2_22_2017.txt was created for 6 sample (ES_2i_Rep1, ES_2i_Rep2, ES_Serum_Rep1, ES_Serum_Rep2, pNPC_Rep1, pNPC_Rep2) experimental analysis. The processed data file Beagan_et_al_YY1_Knockdown_5C_Processed_Data_File_2_22_2017.txt was created for 4 sample (Control_siRNA_Rep1, Control_siRNA_Rep2, YY1_siRNA_Rep1, YY1_siRNA_Rep2) experimental analysis. The raw primer bed file (BED_314-ES-NPC-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). The primer bed file Beagan_et_al_Primer_Bedfile_for_YY1_knockdown.bed provides the same information but only for the primers utilized for the analysis of the control and YY1 siRNA replicates. The binned primer bed files (Beagan_et_al_Binned_Bedfile_2_22_17.bed,Beagan_et_al_YY1_knockdown_Binned_Bedfile_2_22_17.bed) contain 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). The raw counts files (ES_2i_Rep1.counts, ES_2i_Rep2.counts, ES_Serum_Rep1.counts, ES_Serum_Rep2.counts, pNPC_Rep1.counts, pNPC_Rep2.counts, Control_siRNA_Rep1_5C.counts, Control_siRNA_Rep2_5C.counts, YY1_siRNA_Rep1_5C.counts, YY1_siRNA_Rep2_5C.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). Only Sox2 and Klf4 were queried for the siRNA experiments. The raw counts files (ES_2i_Rep1.counts, ES_2i_Rep2.counts, ES_Serum_Rep1.counts, ES_Serum_Rep2.counts, pNPC_Rep1.counts, pNPC_Rep2.counts, Control_siRNA_Rep1_5C.counts, Control_siRNA_Rep2_5C.counts, YY1_siRNA_Rep1_5C.counts, YY1_siRNA_Rep2_5C.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), “Co
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| Submission date |
Feb 27, 2017 |
| 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) |
| GSE85185 |
YY1 and CTCF Orchestrate a 3D Chromatin Looping Switch during Early Neural Lineage Commitment |
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| Relations |
| SRA |
SRX2596663 |
| BioSample |
SAMN06466543 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM2514228_Control_siRNA_Rep2_5C.counts.txt.gz |
149.6 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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