UCSC hub GGR_iter0

ChIP-seq

GGR_ChIP_seq_iter0:

Consists of four subtracks:

• GGR_ChIP_seq_raw_iter0
• GGR_ChIP_seq_norm_iter0
• GGR_ChIP_seq_fragment_extended_iter0
• GGR_ChIP_seq_fragment_extended_norm_iter0

ChIP_seq_raw_iter0:

• Sequencing by Illumina HiSeq
• Raw 50-bp paired-end reads were trimmed using Trimmomatic (v.0.32) (Bolger et al. 2014; doi:10.1093/bioinformatics/btu170).
• Trimmed reads were mapped to human reference genome assembly GRCh38 using bowtie (v.0.12.9) (Li and Durbin 2009; doi:10.1093/bioinformatics/btp324) with options -m 1 -v 2 --best --strata
• PCR duplicates were filtered using Picard tools, http://picard.sourceforge.net
• ENCODE blacklist regions were filtered using bedtools (Quilan and Hall 2010; doi: 10.1093/bioinformatics/btq033)

ChIP_seq_norm_iter0:

• Same as ChIP_seq_raw_iter0 above, except:
• Mappings were normalized by RPKM as implemented in deepTools after extending reads 200 bp in the 3' direction and summing counts in 50-bp bins (Ramirez et al. 2014; doi: 10.1093/nar/gku365).

ChIP_seq_fragment_extended_iter0:

• Same as ChIP_seq_raw_iter0 above, except:
• Reads were extended in 3' direction by the fragment length estimated using SPP (v.2.0) (Karchenko et al. 2008; doi:10.1038/nbt.1508)

ChIP_seq_fragment_extended_norm_iter0:

• Same as ChIP_seq_fragment_extended_iter0 above, except:
• Read mappings were scaled by a constant factor divided by the total number of mapped reads

GGR_ChIP_seq_iter0.peaks:

The tracks in this hub are the same as the tracks in GGR_ChIP_seq_iter0, except with peaks indicated by stacked bedGraph. Peaks were called within-replicate using macs2 (v.2.1.0.20151222)(Zhang et al. 2008; doi: 10.1186/gb-2008-9-9-r137) with parameters --nomodel --extsize $EXTSIZE -g hs -q 0.05 where $EXTSIZE was the fragment length estimated by SPP (v.2.0) (Karchenko et al. 2008; doi:10.1038/nbt.1508). The peaks were then merged across replicates to yield a union set, which is the set shown in this hub. Consists of four subtracks:

• GGR_ChIP_seq_raw_iter0.peaks
• GGR_ChIP_seq_norm_iter0.peaks
• GGR_ChIP_seq_fragment_extended_iter0.peaks
• GGR_ChIP_seq_fragment_extended_norm_iter0.peaks

GGR_ChIP_seq_iter0.peaks_mean:

This track is the same as GGR_ChIP_seq_fragment_extended_norm_iter0.peaks above, except the mean of read mappings was taken across replicates and within timepoints.

GGR_ChIP_seq_iter0.BCL3.diff_sites:

The tracks in this hub are the same as GGR_ChIP_seq_iter0.peaks, except peaks are colored by their direction (increased, decreased, or increased and decreased binding) and strength of differential binding (FDR = 0.01, 0.05 0.1 0.2) across the time course. Differential binding analyses were performed using the negative binomial model implemented in edgeR (Robinson et al. 2010; doi: 10.1093/bioinformatics/btp616). We controlled for batch effects by incorporating all significant surrogate variables estimated using SVAseq (Leek 2014; doi: 10.1093/nar/gku864) into the model as covariates. Normalization factors were computed based on total mapped library size. Consists of four subtracks:

• GGR_ChIP_seq_raw_iter0.BCL3.diff_sites
• GGR_ChIP_seq_norm_iter0.BCL3.diff_sites
• GGR_ChIP_seq_fragment_extended_iter0.BCL3.diff_sites
• GGR_ChIP_seq_fragment_extended_norm_iter0.BCL3.diff_sites

GGR_ChIP_seq_iter0.CEBPB.diff_sites:

Same as GGR_ChIP_seq_iter0.BCL3.diff_sites, except CEBPB instead of BCL3.

GGR_ChIP_seq_iter0.EP300.diff_sites:

Same as GGR_ChIP_seq_iter0.BCL3.diff_sites, except EP300 instead of BCL3.

GGR_ChIP_seq_iter0.FOSL2.diff_sites:

Same as GGR_ChIP_seq_iter0.BCL3.diff_sites, except FOSL2 instead of BCL3.

GGR_ChIP_seq_iter0.HES2.diff_sites:

Same as GGR_ChIP_seq_iter0.BCL3.diff_sites, except HES2 instead of BCL3.

GGR_ChIP_seq_iter0.BCL3.diff_sites_mean:

This track is the same as GGR_ChIP_seq_fragment_extended_norm_iter0.BCL3.diff_sites above, except the mean of read mappings was taken across replicates and within timepoints.

GGR_ChIP_seq_iter0.CEBPB.diff_sites_mean:

Same as GGR_ChIP_seq_iter0.BCL3.diff_sites_mean, except CEBPB instead of BCL3.

GGR_ChIP_seq_iter0.EP300.diff_sites_mean:

Same as GGR_ChIP_seq_iter0.BCL3.diff_sites_mean, except EP300 instead of BCL3.

GGR_ChIP_seq_iter0.FOSL2.diff_sites_mean:

Same as GGR_ChIP_seq_iter0.BCL3.diff_sites_mean, except FOSL2 instead of BCL3.

GGR_ChIP_seq_iter0.HES2.diff_sites_mean:

Same as GGR_ChIP_seq_iter0.BCL3.diff_sites_mean, except HES2 instead of BCL3.

GGR_ChIP_seq_diff_peaks_iter0:

This track contains all differential (and non-differential) binding peaks colored by their direction (increased, decreased, or increased and decreased binding) and strength of differential binding (FDR = 0.01, 0.05 0.1 0.2) across the time course. Presently includes BCL3, CEBPB, EP300, FOSL2, and HES2.

GGR_ChIP_seq_motifs_in_peaks_iter0:

This track contains all binding peaks (union across time course, as previously in GGR_ChIP_seq_iter0.peaks) colored by the strength of their motif match to their consensus binding motif. To search for de novo motifs, the top 500 binding sites by total normalized density were subsetted and the sequences were trimmed to the central 200 bp. MEME (v.4.10.0) was run with the parameters -mod zoops -dna -minw 5 -maxw 12 -nmotifs 20 in order to search for motifs with zero or one occurrence in each sequence with widths between 5 and 12 bp (Bailey et al. 2015; doi: 10.1093/nar/gkv416). Presently includes CEBPB, FOSL2, and GR.

DNase-seq

GGR_DNase_seq_iter0:

Consists of four subtracks:

• GGR_DNase_seq_raw_iter0
• GGR_DNase_seq_norm_iter0
• GGR_DNase_seq_fragment_extended_iter0
• GGR_DNase_seq_fragment_extended_norm_iter0

DNase_seq_raw_iter0:

• Sequencing by Illumina HiSeq
• After stripping custom barcodes, raw reads were mapped with bowtie (v.0.12.9) (Langmead et al. 2009; doi:10.1186/gb-2009-10-3-r25) with options --trim3 30 --seedlen 20 --seedmms 1 -m 1 --best --strata.
• Mappings were filtered for ENCODE blacklisted regions.
• Mappings were filtered for PCR artifacts using a custom python script.
• ENCODE blacklist regions were filtered using bedtools (Quilan and Hall 2010; doi: 10.1093/bioinformatics/btq033)

DNase_seq_norm_iter0:

• Same as DNase_seq_raw_iter0 above, except:
• Mappings were normalized by RPKM as implemented in deepTools after extending reads 200 bp in the 3' direction and summing counts in 50-bp bins (Ramirez et al. 2014; doi: 10.1093/nar/gku365).

DNase_seq_fragment_extended_iter0:

• Same as DNase_seq_raw_iter0 above, except:
• Reads were shifted 100 bp in 3' direction and extended by 200 bp in the 5' direction.

DNase_seq_fragment_extended_norm_iter0:

• Same as DNase_seq_fragment_extended_iter0 above, except:
• Read mappings were scaled by a constant factor divided by the total number of mapped reads

GGR_DNase_seq_iter0.diff_sites:

The tracks in this hub are the same as GGR_DNase_seq_iter0, except with DNase-seq hypersensitive sites (DHSs) colored by their direction (increased, decreased, or increased and decreased accessibility) and strength of differential binding (FDR = 0.01, 0.05 0.1 0.2) across the time course. DHSs were called across replicates and within time point using macs2 (v.2.1.0.20151222)(Zhang et al. 2008; doi: 10.1186/gb-2008-9-9-r137) with parameters -q 0.10 --nomodel --shift -100 --ext 200. Differential accessibility analysis was performed as differential binding analysis, except normalization factors were computed based on number of reads mapped to DHSs rather than total mapped library size. Consists of four subtracks:

• GGR_DNase_seq_raw_iter0.diff_sites
• GGR_DNase_seq_norm_iter0.diff_sites
• GGR_DNase_seq_fragment_extended_iter0.diff_sites
• GGR_DNase_seq_fragment_extended_norm_iter0.diff_sites

GGR_DNase_seq_iter0.diff_sites_mean:

This track is the same as GGR_DNase_seq_fragment_extended_norm_iter0.diff_sites above, except the mean of read mappings was taken across replicates and within timepoints.

GGR_DNase_seq_diff_peaks_iter0:

This track contains all differential (and non-differential) DHSs colored by their direction (increased, decreased, or increased and decreased accessibility) and strength of differential accessibility (FDR = 0.01, 0.05 0.1 0.2) across the time course.

RNA-seq

GGR_RNA_seq_iter0:

Consists of four subtracks:

• GGR_RNA_seq_raw_iter0
• GGR_RNA_seq_norm_iter0
• GGR_RNA_seq_fragment_extended_iter0
• GGR_RNA_seq_fragment_extended_norm_iter0

RNA_seq_raw_iter0:

• Sequencing by Illumina HiSeq
• Raw 50-bp paired-end reads were trimmed using Trimmomatic (v.0.32) (Bolger et al. 2014; doi:10.1093/bioinformatics/btu170).
• Trimmed reads were mapped to human reference genome assembly GRCh38 using STAR aligner (v.2.4.1a) (Dobin et al. 2012: doi:10.1093/bioinformatics/bts635) using the 2-pass procedure wherein novel splice junctions discovered on a first pass of alignments are compiled into a splice junction database used to inform a second pass of alignments as previously described (Engstrom et al. 2013; doi:10.1038/nmeth.2722). STAR aligner was run using default setting except with filtering for non-canonical novel intron junctions and with "sjdbOverhang" parameter set to read length - 1.

RNA_seq_norm_iter0:

• Same as RNA_seq_raw_iter0 above, except:
• Mappings were normalized by RPKM as implemented in deepTools (Ramirez et al. 2014; doi: 10.1093/nar/gku365). (Ramirez et al. 2014; doi: 10.1093/nar/gku365).

GGR_RNA_seq_iter0.diff_sites:

The tracks in this hub are the same as GGR_RNA_seq_iter0, except with differentially expressed genes (DEGs) colored by their direction (increased, decreased, or increased and decreased expression) and strength of differential expression (FDR = 0.01, 0.05 0.1 0.2) across the time course. RNA-seq read counts were quantified in gene body exons as annotated in GENCODE (v.22) (Harrow et al. 2012; doi: 10.1101/gr.135350.111) using featureCounts (v.1.4.6-p4) (Liao et al. 2014; doi: 10.1093/bioinformatics/btt656) where both paired reads were required to map to the same gene to be counted. Differential expression analysis was performed in the same manner as the differential accessibility analysis.

• GGR_RNA_seq_raw_iter0.diff_sites
• GGR_RNA_seq_norm_iter0.diff_sites
• GGR_RNA_seq_fragment_extended_iter0.diff_sites
• GGR_RNA_seq_fragment_extended_norm_iter0.diff_sites

GGR_RNA_seq_degs_iter0:

This track contains all DEGs (and tested non-DEGs) colored by their direction (increased, decreased, or increased and decreased expression) and strength of differential expression (FDR = 0.01, 0.05 0.1 0.2) across the time course.