=> see bash code below for the size of the tar files (to enter manually)

• Introduction: Quick Start, Tutorial, Flowchart, Outputs structure
• Install: Dependencies, Containers, References, Test datasets
• Inputs: Data, Design, Parameters
• 1. Preprocessing: ATAC reads, ATAC peaks, mRNA
• 2. Differential Analysis: ATAC, mRNA, Split
• 3. Enrichment Analysis: Enrichment, Figures, Tables

Introduction

Tests datasets have beed generated for each of the 4 species supported by Cactus. This allows to make sure the pipeline works equally well for each species. Downloading the smallest datasets (fly or worm) should be sufficient for users to get a sense on how the pipeline works. Here are the sizes of the test datasets:

specie	raw_files	sampled_files	sampled_atac	sampled_mrna	tar
fly	17 GB	377 MB	329 MB	48 MB	360 MB
worm	68 GB	1.4 GB	1.3 GB	41 MB	1.3 GB
mouse	52 GB	6 GB	5.9 GB	142 MB	5.7 GB
human	118 GB	9 GB	8.8 GB	190 MB	8.5 GB

NOTE: Sampled mRNA-Seq datasets are similar between the 4 species, however, sampled ATAC-Seq datasets are much larger for human and mice than for worm and fly. This is due to large difference in genome sizes but not in transcriptome size between these species; as shown here:

species	genome	transcriptome
fly	100 MB	53 MB
worm	144 MB	89 MB
human	2731 MB	158 MB
mouse	3100 MB	261 MB

Below is a table that shows the time needed to run each tests datasets on a local server with 47 CPUs and 300Gb of RAM:

Species	Duration	CPU_hours	Tasks
fly	22m	9.8	1496
worm	11m	4.2	874
human	2h 12m	51.5	981
mouse	3h 27m	77.5	801

Downloading test datasets

The test datasets can be downloaded with this command:

nextflow run jsalignon/cactus/scripts/download/download.nf --test_datasets --species worm -r main -latest

The parameters for this command are:

• --species: can be any of the 4 species supported by Cactus (worm, fly, mouse or human)
• --threads can be set to determine the number of thread used by pigz for uncompressing the references archive files

NOTE: The test datasets contains 3 folders: data, design and parameters; as described in the Inputs section.

NOTE: A template script to run all test datasets with all tools manager can be found here.

Details on the test datasets origins and labels

Worm and human (GSE98758)

GEO Status: Public on Aug 29, 2018

GEO Title: Genome-wide DNA accessibility maps and differential gene expression using ChIP-seq, ATAC-seq and RNA-seq for the human secondary fibroblast cell line hiF-T and whole worms with and without knockdown of FACT complex

GEO Summary: To assess the mechanisms by which FACT depletion leads to increased sensitivity of cells to be reprogrammed, we measured the chromatin accessibility landscape using ATAC-seq following mock treatment, SSRP1 knockdown, or SUPT16H knockdown in human fibroblasts and mock, hmg-3 or hmg-4 knockdown in whole worms, and differential gene expression in hmg-3 knockout mutants or following mock, hmg-4, or spt-16 knockdown by RNAseq.

GEO Design: Examination of two FACT complex components in human cells and worms with ChIP-seq, ATAC-seq and RNA-seq

Citation: Kolundzic E, Ofenbauer A, Bulut SI, Uyar B et al. FACT Sets a Barrier for Cell Fate Reprogramming in Caenorhabditis elegans and Human Cells. Dev Cell 2018 Sep 10;46(5):611-626.e12. PMID: 30078731

Abstract: The chromatin regulator FACT (facilitates chromatin transcription) is essential for ensuring stable gene expression by promoting transcription. In a genetic screen using Caenorhabditis elegans, we identified that FACT maintains cell identities and acts as a barrier for transcription factor-mediated cell fate reprogramming. Strikingly, FACT’s role as a barrier to cell fate conversion is conserved in humans as we show that FACT depletion enhances reprogramming of fibroblasts. Such activity is unexpected because FACT is known as a positive regulator of gene expression, and previously described reprogramming barriers typically repress gene expression. While FACT depletion in human fibroblasts results in decreased expression of many genes, a number of FACT-occupied genes, including reprogramming-promoting factors, show increased expression upon FACT depletion, suggesting a repressive function of FACT. Our findings identify FACT as a cellular reprogramming barrier in C. elegans and humans, revealing an evolutionarily conserved mechanism for cell fate protection.

Homologs:

Worm	Humans
hmg-4, hmg-3	SSRP1
spt-16	SUPT16H

Worm samples:

sample_id	library_strategy	sample_title	srr_id	gsm_id	library_layout
input	ATAC-seq	input_control	SRR5000684	GSM2385318	PAIRED
ctl_1	RNA-Seq	ctrl_rep1	SRR5860412	GSM2715402	SINGLE
ctl_2	RNA-Seq	ctrl_rep2	SRR5860413	GSM2715403	SINGLE
ctl_3	RNA-Seq	ctrl_rep3	SRR5860414	GSM2715404	SINGLE
hmg4_1	RNA-Seq	hmg4_rep1	SRR5860415	GSM2715405	SINGLE
hmg4_2	RNA-Seq	hmg4_rep2	SRR5860416	GSM2715406	SINGLE
hmg4_3	RNA-Seq	hmg4_rep3	SRR5860417	GSM2715407	SINGLE
spt16_1	RNA-Seq	spt16_rep1	SRR5860418	GSM2715408	SINGLE
spt16_2	RNA-Seq	spt16_rep2	SRR5860419	GSM2715409	SINGLE
spt16_3	RNA-Seq	spt16_rep3	SRR5860420	GSM2715410	SINGLE
ctl_1	ATAC-seq	Rluc_rep1	SRR5860424	GSM2715414	PAIRED
ctl_2	ATAC-seq	Rluc_rep2	SRR5860425	GSM2715415	PAIRED
ctl_3	ATAC-seq	Rluc_rep3	SRR5860426	GSM2715416	PAIRED
spt16_1	ATAC-seq	spt-16_rep1	SRR5860430	GSM2715420	PAIRED
spt16_2	ATAC-seq	spt-16_rep2	SRR5860431	GSM2715421	PAIRED
spt16_3	ATAC-seq	spt-16_rep3	SRR5860432	GSM2715422	PAIRED
hmg4_1	ATAC-seq	hmg-4_rep1	SRR5860433	GSM2715423	PAIRED
hmg4_2	ATAC-seq	hmg-4_rep2	SRR5860434	GSM2715424	PAIRED
hmg4_3	ATAC-seq	hmg-4_rep3	SRR5860435	GSM2715425	PAIRED

Human samples:

sample_id	library_strategy	sample_title	srr_id	gsm_id	library_layout
ctl_1	ATAC-seq	mock_ATAC-seq_rep1	SRR5521292	GSM2611319	PAIRED
ctl_2	ATAC-seq	mock_ATAC-seq_rep2	SRR5521293	GSM2611320	PAIRED
ssrp1_1	ATAC-seq	SSRP1_ATAC-seq_rep1	SRR5521294	GSM2611321	PAIRED
ssrp1_2	ATAC-seq	SSRP1_ATAC-seq_rep2	SRR5521295	GSM2611322	PAIRED
supt16h_1	ATAC-seq	SUPT16H_ATAC-seq_rep1	SRR5521296	GSM2611323	PAIRED
supt16h_2	ATAC-seq	SUPT16H_ATAC-seq_rep2	SRR5521297	GSM2611324	PAIRED
ctl_1	RNA-Seq	pri_mockTotal_A:_human_mock_RNA-seq_rep1	SRR7101006	GSM3127942	PAIRED
ctl_2	RNA-Seq	pri_mockTotal_B:_human_mock_RNA-seq_rep2	SRR7101007	GSM3127943	PAIRED
ctl_3	RNA-Seq	pri_mockTotal_C:_human_mock_RNA-seq_rep3	SRR7101008	GSM3127944	PAIRED
ssrp1_1	RNA-Seq	pri_ssrp1Total_A:_human_Ssrp1_kd_RNA-seq_rep1	SRR7101009	GSM3127945	PAIRED
ssrp1_2	RNA-Seq	pri_ssrp1Total_B:_human_Ssrp1_kd_RNA-seq_rep2	SRR7101010	GSM3127946	PAIRED
ssrp1_3	RNA-Seq	pri_ssrp1Total_C:_human_Ssrp1_kd_RNA-seq_rep3	SRR7101011	GSM3127947	PAIRED
supt16h_1	RNA-Seq	pri_supt16hTotal_A:_human_Supt16h_kd_RNA-seq_rep1	SRR7101012	GSM3127948	PAIRED
supt16h_2	RNA-Seq	pri_supt16hTotal_B:_human_Supt16h_kd_RNA-seq_rep2	SRR7101013	GSM3127949	PAIRED
supt16h_3	RNA-Seq	pri_supt16hTotal_C:_human_Supt16h_kd_RNA-seq_rep3	SRR7101014	GSM3127950	PAIRED

Fly (GSE149339)

GEO Status: Public on May 10, 2020

GEO Title: Pioneer factor GAF cooperates with PBAP and NURF to regulate transcription

GEO Summary: The Drosophila pioneer factor GAF is known to be essential for RNA Pol II promoter-proximal pausing and the removal of nucleosomes from a set of target promoters with GAGAG motifs. We and others have speculated that GAF recruits the ISWI family ATP-dependent chromatin remodeling complex NURF, on the basis that NURF and GAF are both required to remodel nucleosomes on an hsp70 promoter in vitro and that GAF interacts physically with NURF. However, GAF was also recently shown to interact with PBAP, a SWI/SNF family remodeler. To test which of these remodeling complexes GAF works with, we depleted GAF, NURF301, BAP170, and NURF301+BAP170 in Drosophila S2 cells using RNAi. We used a combination of PRO-seq, ATAC-seq, 3'RNA-seq, and CUT&RUN to demonstrate that while GAF and PBAP synergistically open chromatin at target promoters which allows Pol II recruitment and pausing to proceed, GAF and NURF also synergistically position the +1 nucleosome to ensure efficient pause release and transition to productive elongation.

GEO Design: We treated two independent replicates of Drosophila S2 cells with dsRNA to LACZ (control), GAF, NURF301, BAP170 (the unique subunits of the NURF and PBAP complexes, respectively), and NURF301+BAP170. After 5 days, we harvested cells, validated knockdowns, and performed PRO-seq, ATAC-seq and 3'RNA-seq. We also performed CUT&RUN for both GAF and NURF301 in untreated S2 cells.

Citation: Judd, J., Duarte, F. M. & Lis, J. T. Pioneer-like factor GAF cooperates with PBAP (SWI/SNF) and NURF (ISWI) to regulate transcription. Genes Dev. 35, 147–156 (2021).

Abstract: Transcriptionally silent genes must be activated throughout development. This requires nucleosomes be removed from promoters and enhancers to allow transcription factor (TF) binding and recruitment of coactivators and RNA polymerase II (Pol II). Specialized pioneer TFs bind nucleosome-wrapped DNA to perform this chromatin opening by mechanisms that remain incompletely understood. Here, we show that GAGA factor (GAF), a Drosophila pioneer-like factor, functions with both SWI/SNF and ISWI family chromatin remodelers to allow recruitment of Pol II and entry to a promoter-proximal paused state, and also to promote Pol II's transition to productive elongation. We found that GAF interacts with PBAP (SWI/SNF) to open chromatin and allow Pol II to be recruited. Importantly, this activity is not dependent on NURF as previously proposed; however, GAF also synergizes with NURF downstream from this process to ensure efficient Pol II pause release and transition to productive elongation, apparently through its role in precisely positioning the +1 nucleosome. These results demonstrate how a single sequence-specific pioneer TF can synergize with remodelers to activate sets of genes. Furthermore, this behavior of remodelers is consistent with findings in yeast and mice, and likely represents general, conserved mechanisms found throughout eukarya.

Samples:

sample_id	library_strategy	sample_title	srr_id	gsm_id	library_layout
ctl_1	ATAC-seq	LACZ_ATACseq_Rep1	SRR11607688	GSM4498282	PAIRED
ctl_1	ATAC-seq	LACZ_ATACseq_Rep1	SRR11607689	GSM4498282	PAIRED
ctl_2	ATAC-seq	LACZ_ATACseq_Rep2	SRR11607690	GSM4498283	PAIRED
ctl_2	ATAC-seq	LACZ_ATACseq_Rep2	SRR11607691	GSM4498283	PAIRED
gaf_1	ATAC-seq	GAF_ATACseq_Rep1	SRR11607692	GSM4498284	PAIRED
gaf_1	ATAC-seq	GAF_ATACseq_Rep1	SRR11607693	GSM4498284	PAIRED
gaf_2	ATAC-seq	GAF_ATACseq_Rep2	SRR11607675	GSM4498285	PAIRED
gaf_2	ATAC-seq	GAF_ATACseq_Rep2	SRR11607694	GSM4498285	PAIRED
b170_1	ATAC-seq	BAP170_ATACseq_Rep1	SRR11607676	GSM4498286	PAIRED
b170_1	ATAC-seq	BAP170_ATACseq_Rep1	SRR11607677	GSM4498286	PAIRED
b170_2	ATAC-seq	BAP170_ATACseq_Rep2	SRR11607678	GSM4498287	PAIRED
b170_2	ATAC-seq	BAP170_ATACseq_Rep2	SRR11607679	GSM4498287	PAIRED
n301_1	ATAC-seq	NURF301_ATACseq_Rep1	SRR11607680	GSM4498288	PAIRED
n301_1	ATAC-seq	NURF301_ATACseq_Rep1	SRR11607681	GSM4498288	PAIRED
n301_2	ATAC-seq	NURF301_ATACseq_Rep2	SRR11607682	GSM4498289	PAIRED
n301_2	ATAC-seq	NURF301_ATACseq_Rep2	SRR11607683	GSM4498289	PAIRED
n301b170_1	ATAC-seq	NURF301BAP170_ATACseq_Rep1	SRR11607684	GSM4498290	PAIRED
n301b170_1	ATAC-seq	NURF301BAP170_ATACseq_Rep1	SRR11607685	GSM4498290	PAIRED
n301b170_2	ATAC-seq	NURF301BAP170_ATACseq_Rep2	SRR11607686	GSM4498291	PAIRED
n301b170_2	ATAC-seq	NURF301BAP170_ATACseq_Rep2	SRR11607687	GSM4498291	PAIRED
gaf_2	RNA-Seq	GAF_RNAseq_Rep2	SRR11607698	GSM4498295	SINGLE
b170_1	RNA-Seq	BAP170_RNAseq_Rep1	SRR11607699	GSM4498296	SINGLE
b170_2	RNA-Seq	BAP170_RNAseq_Rep2	SRR11607700	GSM4498297	SINGLE
n301_1	RNA-Seq	NURF301_RNAseq_Rep1	SRR11607701	GSM4498298	SINGLE
n301_2	RNA-Seq	NURF301_RNAseq_Rep2	SRR11607702	GSM4498299	SINGLE
n301b170_1	RNA-Seq	NURF301BAP170_RNAseq_Rep1	SRR11607703	GSM4498300	SINGLE
n301b170_2	RNA-Seq	NURF301BAP170_RNAseq_Rep2	SRR11607704	GSM4498301	SINGLE
ctl_1	RNA-Seq	LACZ_RNAseq_Rep1	SRR11607695	GSM4498292	SINGLE
ctl_2	RNA-Seq	LACZ_RNAseq_Rep2	SRR11607696	GSM4498293	SINGLE
gaf_1	RNA-Seq	GAF_RNAseq_Rep1	SRR11607697	GSM4498294	SINGLE

Mouse (GSE193393)

GEO Status: Public on Jun 23, 2022

GEO Title: PHF20 Activates Autophagy Genes through Enhancer Activation via H3K36me2 Binding Activity

GEO Summary: Autophagy is a catabolic pathway that maintains cellular homeostasis under various stress conditions, including nutrient-deprived conditions. To elevate autophagic flux to a sufficient level under stress conditions, transcriptional activation of autophagy genes occurs to replenish autophagy components. Here, using combination of RNA-seq, ATAC-seq and ChIP-seq, we demonstrated found that plant homeodomain finger protein 20 (Phf20PHF20), which is an epigenetic reader possessing methyl binding activity, plays a key role in controlling the expression of autophagy genes. PHF20 activates autophagy genes through enhancer activation via H3K36me2 binding activity as an epigenetic reader and that our findings emphasize the importance of nuclear regulation of autophagy.

GEO Design: mRNA-seq, ATAC-seq and ChIP-seq experiments under normal and 24hrs of glucose starvation condition in WT and Phf20-/- MEFs

Citation: Park SW, Kim J, Oh S, Lee J et al. PHF20 is crucial for epigenetic control of starvation-induced autophagy through enhancer activation. Nucleic Acids Res 2022 Aug 12;50(14):7856-7872. PMID: 35821310

Samples:

sample_id	library_strategy	sample_title	srr_id	gsm_id	library_layout
Wt_1	ATAC-seq	WT_control_ATAC-seq_rep1	SRR17483668	GSM5776726	PAIRED
Wt_2	ATAC-seq	WT_control_ATAC-seq_rep2	SRR17483667	GSM5776727	PAIRED
WtStarv_1	ATAC-seq	WT_GlcStarv_ATAC-seq_rep1	SRR17483666	GSM5776728	PAIRED
WtStarv_2	ATAC-seq	WT_GlcStarv_ATAC-seq_rep2	SRR17483665	GSM5776729	PAIRED
Phf_1	ATAC-seq	Phf20-/-_control_ATAC-seq_rep1	SRR17483664	GSM5776730	PAIRED
Phf_2	ATAC-seq	Phf20-/-_control_ATAC-seq_rep2	SRR17483663	GSM5776731	PAIRED
PhfStarv_1	ATAC-seq	Phf20-/-_GlcStarv_ATAC-seq_rep1	SRR17483662	GSM5776732	PAIRED
PhfStarv_2	ATAC-seq	Phf20-/-_GlcStarv_ATAC-seq_rep2	SRR17483661	GSM5776733	PAIRED
Wt_1	RNA-Seq	WT_control_RNA-seq_rep1	SRR17535397	GSM5799507	PAIRED
Wt_2	RNA-Seq	WT_control_RNA-seq_rep2	SRR17535396	GSM5799508	PAIRED
WtStarv_1	RNA-Seq	WT_GlcStarv_RNA-seq_rep1	SRR17535395	GSM5799509	PAIRED
WtStarv_2	RNA-Seq	WT_GlcStarv_RNA-seq_rep2	SRR17535394	GSM5799510	PAIRED
Phf_1	RNA-Seq	Phf20-/-_control_RNA-seq_rep1	SRR17535392	GSM5799511	PAIRED
Phf_2	RNA-Seq	Phf20-/-_control_RNA-seq_rep2	SRR17535391	GSM5799512	PAIRED
PhfStarv_1	RNA-Seq	Phf20-/-_GlcStarv_RNA-seq_rep1	SRR17535390	GSM5799513	PAIRED
PhfStarv_2	RNA-Seq	Phf20-/-_GlcStarv_RNA-seq_rep2	SRR17535393	GSM5799514	PAIRED