scsHi-C analysis

This repository contains notebooks for the analysis of scsHi-C data.

Preprocessing

Preprocessing of scsHi-C experiments was done as follows: First, the scshic_pipeline was applied to raw sequencing data. The resulting cooler files were then balanced as follows:

• All-contact coolers were zoomified and balanced conventionally (see the following script for an example for G2 WT data)
• Cis-sister and trans-sister contacts were pooled (see the following script for an example for G2 WT data)
• Then, the pooled cis-and-trans sister contacts were zoomified and balanced (see the following script for an example for G2 WT data)
• Then, cis-sister and trans-sister coolers were seperately zoomified (see the following script for an example for G2 WT data) and the obtained weights from the cis-and-trans sister coolers transferred to the cis-sister and trans-sister file respectively (see the following script for an example for G2 WT data).

Fig. 1

(d)

The analysis of relative mutation rates between a sample grown for 5 days in 4sT and a control sample before and after OsO4-mediated conversion can be found in this notebook.

Fig. 2

(a)

The visualization of the example region on chr.1 for the pooled G2 WT samples can be found in this notebook.

(b)

The visualization of the example region on chr.1 for the pooled Prometaphase samples can be found in this notebook.

(c) and (d)

The calculation of genomie scaling curves of the pooled G2 WT sample and the pooled prometaphase sample can be found in this notebook

(e)

The visualization of the example region on chr.8 for the pooled G2 WT samples can be found in this notebook.

(f), (g) and (h)

The quantification visualization of LOLA-enrichment scores, the quantification of H3K27me3 intensity at highly paired and highly unpaired domains and the stack-up of line-profiles at highly paired and highly-unpaired regions can be found in this notebook.

Fig. 3

(a)

The visualization of the example region on chr.2 for the pooled G2 WT samples as well as the calculation of cis-sister and trans-sister contact density can be found in this notebook.

(b), (e) and (f)

The pileup of cis-sister and trans-sister contacts at TAD-centers as well as the stak-up of line profiles along TADs and the quantification of enrichment of trans-sister contacts at TAD-boundaries can be found in the following notebook.

Fig. 4

(a)

The visualization of the example region on chr. 1 for the G2 WT sample can be found in the following notebook. The visualization of the example region on chr. 1 for the Nipbl-degraded sample can be found in the following notebook. The visualiztion of the example region on chr. 1 for the Sororin-degrade sample can be found in the folloiwing notebook.

(b)

The calculation of genomic scaling plots for the WT sample, the Sorroin-degraded sample and the Nipbl-degraded sample can be found in the following notebook.

(c)

The pileup of TAD-centers for Nipbl-degraded sample can be found in the following notebook. A similar analysis for the Sororin sample can be found here.

(d)

The obs/expected stack-up calculations can be found in the following notebooks: WT, Nipbl, Sororin.

(e)

The quantification of contact amount at TAD-boundaries for G2 WT samples, G2 Nipbl-degraded samples and G2 Sororin-degraded samples as done in this notebook.

Extended Data Fig. 1

(d)

The quantification of percentage of cells that is alive upon treatment with different concentrations of 4sT and etoposide for 24h was done in this notebook

(f)

The quantifiction of normalized (to control) p-gamma-H2A.X signal upon treatment with 4sT and etoposide for 24h was done in this notebook

(i)

The quantification of doubly labelled reads in scsHi-C samples released into S-Phase for different amounts of time can be found in this notebook.

(j)

The quantification of trans-sister contacts in scsHi-C samples generated n G2, prometaphase in the following G1 phase can be found in this notebook.

Extended Data Fig. 2

(a)

Mass spec based quantification of 4sT in DNA from cells grown for 5 days in 4sT was done in the following notebook

(b)

Quantification of sequencing based incorporation of 4sT was done in the following notebook

Extended Data Fig. 3

(a)

Histograms of signature mutations per read were calculated in the following notebook.

(b)

The false-positive rate of doubly labelled read detection was calculated in the following notebook.

(d)

The percentage of doubly labelled reads at different numbers of signature mutations was calculated in this notebook.

(e)

The amount of wrongly assigned trans-sister contacts was calculated in this notebook.

Extended Data Figure 4

(a)

Example regions for two of the G2 WT replicates was visualized in this notebook.

(b)

HiCrep was run for the different G2 WT replicates in this notebook.

(c)

Example regions for the two Prometaphase replicates were visualized in this [notebook] (https://github.com/Mittmich/scsHiCanalysis/blob/master/notebooks/Prometaphase_ind_examples.ipynb).

(d)

HiCrep was run for the different prometaphase replicates in this notebook.

Extneded Data Figure 5

(a)

Example of G2 WT data on chromosome 3 was visualized in the following notebook.

(b)

Correlation of scsHi-C data with FISH loci-splitting frequency was done in the following notebook

(c)

Correlation of scsHi-C data with dCas9 loci splitting frequency was done in the following notebook

(d)

Histogram of average trans-sister contact per TAd was calculated in the following notebook.

(e)

Scaling plots of cis-sister and trans-sister contacts in highly paired and highly unpaired domains was calculated in the following notebook

(f)

Average H3K27me3 signal at high-pairing domains was calculated in the folloiwng notebook.

Extended Data Figure 6

(a), (b), (c)

Example regions in this figure were plotted in the following notebook.

(d)

Hi-C stack-ups at CTCF sites were calculated in the following notebook.

(e)

Quantification of Hi-C signal at CTCF sites was done in the following notebook.

Extended Data Figure 7

(c)

Scaling plots for WT G2 and Nipbl-degraded samples constructed from all reads can be found in the following notebook.

(e)

Example regions were plotted in the following notebook: WT, Sororin-degraded, NIPBL-degraded.

(f)

HiC-rep analysis of all NIPBL-degraded samples was done in the following notebook. HiC-rep analysis of Sororin-degraded samples was done in the following notebook.

Extended Data Figure 8

(a)

ICCF stack up analysis of NIPBL-degraded cells was done in the following notebook.

(b)

Obs/exp stack up at TADs of WT and NIPBL-degraded cells was done in the following notebook.

(c)

ICCF stack up analysis at highly paired and highly unpaired domains was done in the following notebook.

(d)

Pairing score stack up analysis at highly paired and highly unpaired domains was done in the following notebook.