Description

These tracks represent data downloaded from GEO for RNAseq assays from the Weizmann institute uploaded in 2014 August.

Each track indicates the genomic coverage from strand-specific RNAseq data (on either the plus or minus strand) in the mouse mm10 genome assembly for cells in the hematopoetic lineage. Coverage has been normalized between samples as described in Methods below. The names of the samples are as below.

 Sample        Run          cellType        Organ         SelectionMarker                                             
 hscxx_wz380   SRR1536380   (HSC)           Bone Marrow   Lin-, ckit+, Sca1+, Flk2-, CD34+                            
 hscxx_wz382   SRR1536382   (HSC)           Bone Marrow   Lin-, ckit+, Sca1+, Flk2-, CD34+                            
 mppxx_wz383   SRR1536383   (MPP)           Bone Marrow   Lin-, ckit+, Sca1+, Flk2+, CD34+                            
 mppxx_wz384   SRR1536384   (MPP)           Bone Marrow   Lin-, ckit+, Sca1+, Flk2+, CD34+                            
 clpxx_wz387   SRR1536387   (CLP)           Bone Marrow   Lin-, ckit+, Flk2+, Il7R+                                   
 clpxx_wz388   SRR1536388   (CLP)           Bone Marrow   Lin-, ckit+, Flk2+, Il7R+                                   
 cmpxx_wz391   SRR1536391   (CMP)           Bone Marrow   Lin-, ckit+, Sca1-, CD34+, FcgRIII int                      
 cmpxx_wz392   SRR1536392   (CMP)           Bone Marrow   Lin-, ckit+, Sca1-, CD34+, FcgRIII int                      
 gmpxx_wz393   SRR1536393   (GMP)           Bone Marrow   Lin-, ckit+, Sca1-, CD34+, FcgRIII high                     
 gmpxx_wz394   SRR1536394   (GMP)           Bone Marrow   Lin-, ckit+, Sca1-, CD34+, FcgRIII high                     
 macro_wz397   SRR1536397   Macrophages     Bone Marrow   B220-, CD3-, NK1.1-, F4/80+, CD115-, low SSC                
 macro_wz398   SRR1536398   Macrophages     Bone Marrow   B220-, CD3-, NK1.1-, F4/80+, CD115-, low SSC                
 granu_wz401   SRR1536401   Granulocytes    Bone Marrow   B220-, CD3-, NK1.1-, Gr1+, high SSC                         
 granu_wz402   SRR1536402   Granulocytes    Bone Marrow   B220-, CD3-, NK1.1-, Gr1+, high SSC                         
 monoc_wz409   SRR1536409   Monocytes       Bone Marrow   B220-, CD3-, NK1.1-, CD115+, low SSC                        
 monoc_wz410   SRR1536410   Monocytes       Bone Marrow   B220-, CD3-, NK1.1-, CD115+, low SSC                        
 bcell_wz411   SRR1536411   B cells         Spleen        B220+, CD3-, CD19+                                          
 bcell_wz412   SRR1536412   B cells         Spleen        B220+, CD3-, CD19+                                          
 tcd4p_wz415   SRR1536415   T CD4+ cells    Spleen        B220-, CD3+, CD19-, CD4+, CD8-                              
 tcd4p_wz416   SRR1536416   T CD4+ cells    Spleen        B220-, CD3+, CD19-, CD4+, CD8-                              
 tcd8p_wz419   SRR1536419   T CD8+ cells    Spleen        B220-, CD3+, CD19-, CD4-, CD8+                              
 tcd8p_wz420   SRR1536420   T CD8+ cells    Spleen        B220-, CD3+, CD19-, CD4-, CD8+                              
 nkcel_wz421   SRR1536421   NK Cells        Spleen        B220-, CD3-, CD19-, CD4-, CD8-, Terr119-, TCRbeta-, NK1.1+  
 nkcel_wz422   SRR1536422   NK Cells        Spleen        B220-, CD3-, CD19-, CD4-, CD8-, Terr119-, TCRbeta-, NK1.1+  
 mepxx_wz425   SRR1536425   (MEP)           Bone Marrow   Lin-, ckit+, Sca1-, Flk2-, CD34-                            
 mepxx_wz426   SRR1536426   (MEP)           Bone Marrow   Lin-, ckit+, Sca1-, Flk2-, CD34-                            
 eryax_wz427   SRR1536427   (Ery A)         Spleen        B220-, CD3-, Ter119+, CD71+                                 
 eryax_wz428   SRR1536428   (Ery A)         Spleen        B220-, CD3-, Ter119+, CD71+                                 

Display Conventions and Configuration

The minus strand coverage tracks use negative values so that they descend from the zero line. The plus strand coverage tracks use positive values. The colors have been chosen to be colorblind-friendly:

•  Blue  - plus strand coverage
•  Red  - minus strand coverage

Because the coverage values have been normalized between all the samples, the visual display indicates the relative expression between samples at a locus as long as all the individual tracks use the same scale (adjustable with "Vertical viewing range" limits).

Since there is wide variation in coverage between genes with different levels of expression, you should adjust the "Vertical viewing range" control at the composite track level in order to vertically zoom in and out at a given locus. In general, you should probably keep the plus and minus sets of tracks at the same scale. However, you might also want to use different plus and minus scales to more closely examine cases of anti-sense transcription. Although you can adjust each sample's scale separately, this will distort the relative expression of that sample, so should be avoided. If you do this inadvertantly, the "Reset to defaults" function can be used to restore all the individual track settings.

Because the plus and minus strand are aggregated into separate composite tracks, the default browser display groups them separately. Be aware that you can drag the tracks individually to reorder them. For example, you might want to place each sample's plus and minus strands together, with plus above minus for a more natural display.

Methods

Trimming and Filtering

The raw single-end reads were trimmed to eliminate low quality bases and so that the data for all samples would consist of 42bp reads. The trimmed reads were mapped with Bowtie2 (Langmead et al., 2012) to a set of repeat-elements for the appropriate species. Reads mapping to these elements were removed from further processing.

Alignment and Duplicate Removal

The filtered reads were aligned to the appropriate genome assembly with STAR (Dobin et al., 2012) keeping only the primary mapping for multiply-mapped reads. Duplicate mappings were removed with Samtools.

Coverage and Normalization

The duplicate-removed alignments were converted to coverage using bedTools. The count of mapped reads after duplicate removal in each sample was used to normalize the coverage across samples. These counts were converted to "sizeFactor" values whose product is one, in the same manner as is done in DESEQ2 (Love et al., 2014). This normalization compensates for differences in sequencing depth between the samples. The un-normalized values are divided by the sizeFactors before the rest of the DESEQ2 algorithm is performed. In the same way, the coverage values from bedTools have been divided by the sizeFactor values to create the tracks presented here.

References

Lara-Astiaso D, Weiner A, Lorenzo-Vivas E, Zaretsky I et al. Immunogenetics. Chromatin state dynamics during blood formation. Science 2014 Aug 22;345(6199):943-9. PMID: 25103404

Langmead, B., and Salzberg, S. (2012). Fast gapped-read alignment with Bowtie 2. Nature Methods 9, 357-359.

Dobin, A., Davis, C.A., Schlesinger, F., Drenkow, J., Zaleski, C., Jha, S., Batut, P., Chaisson, M., and Gingeras, T.R. (2013). STAR: ultrafast universal RNA-seq aligner. Bioinformatics 29(1), 15-21.

Love, M.I., Huber, W., and Anders, S. (2014). Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biology, 15, 550.