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Test_datasets.md

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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