title: SAMPLE 24: wt3 60min

city: Geneva

first: Natacha

last: Civic

address: Rue Michel-Servet 1

department: iGE3 Genomics Platform

country: Switzerland

institute: University of Geneva

hyb_protocol: 50 ng of RNA were hybridized at 65°C over night

scan_protocol: Hybridization mixes were then loaded in the nCounter Prep Station for purification of complexes and loading on the cartridge. Cartridges were scanned on the nCounter Flex scanner.

data_processing: according to J Microbiol Methods. 2011 Feb;84(2):327-34

label: direct hybridization with molecular fluorecent barcode probes

organism: Pseudomonas aeruginosa PAO1

source_name: wt culture 3; 60 min after Zn treatment

label_protocol: no labelling - Direct hybridization of the RNA with the nanostring codeset (mix of probes)

growth_protocol: Cultures were grown at 37°C in modified Luria-Broth medium as described in DOI: 10.3389/fmicb.2021.739988

extract_protocol: For each time point, 0,5ml of culture was mixed with 1ml of RNA protect Reagent. Total RNAs were then extracted with RNeasy mini kit (Qiagen) according to the manufacturer'sinstructions and treated withRQ+ RNAse-free DNAse for 2h at 37°C, followd by phenol/chloroform extraction and ethanol precipitation. 50ng of total RNA were used for NanoString nCounter expression analysis.

value: PAO1 WT Laboratory strain

value: zinc induction


title: HNF6-KO_ESC_Rep2

status: Public on Oct 27 2021

type: SRA

city: Aachen

first: Ivan

last: Costa

address: Pauwelsstraße 19

country: Germany

institute: Unikilink RWTH Aachen

library_source: transcriptomic

data_processing:
HiSeq 3000/4000 System (Illumina®) with single-end reads of 50bp
Reads were aligned with STAR aligner (Dobin, 2013; Version 2.5.2b) on  human genome hg38 and using Ensembl Transcriptome Annotation e87 as  transcriptome reference.
DESeq2 (version 1.22.1) was used for  normalization and differential expression analysis (Love, 2014). Only  genes with at least 10 reads, an adjusted p-value < 0.05 and abs(FC) >  1.5 were considered
SAMtools and deepTools were used for further processing
Genome_build: hg38
Supplementary_files_format_and_content: We provide bigwig, VST(Variance Stabilizing Transformation) model count and FPKM(Fragments Per Kilobase Million)

molecule: total RNA

organism: Homo sapiens

growth_protocol: HUES8 control and HNF6 KO hESC were subjected to in vitro pancreatic differentiation.

extract_protocol:
Cells were harvested at definitve endoderm (DE), pancreatic endoderm (PE) and pancreatic progenitor (PP) stage. Total RNA from ESC-derived pancreatic cells was isolated using GeneJET RNA Purification Kit (Thermo Fisher Scientific) according to manufacturer’s instructions.
After quality check with samples reaching RNA integrity values RIN>8, up to 1µg of total RNA was used for poly-A enrichment and subsequent library preparation with the TrueSeq stranded mRNA Kit from Illumina (HUES8 n=6)

tag: tissue

value: embryonic stem cells, HUES8

tag: genotype

tag: Stage

value: undifferentiated


title: Con_PDX1-NKX6.1-pos_Rep3

value: wild type

value: pancreatic progenitors, sorted PDX1+/NKX6.1+


title: P30WTnt1

city: New York

first: Jens

last: Hansen

state: NY

address: 1425 Madison Ave

department: Department of Pharmacological Sciences

country: USA

institute: Icahn School of Medicine at Mount Sinai

data_processing:
Random removal of reads from each fastq-file to ensure that all samples that are subjected to the same comparison have the same final read counts (e.g. all mouse samples have the same read counts before alignment)
Alignment to the reference genome using Tophat 2.0.8
Identification of differentially expressed genes using Cufflinks 1.3.0
Genome_build: h19 and mm9

organism: Mus musculus

source_name: Thoracic aortic wall

extract_protocol: none provided by the submitter

value: thoracic aortic tissue from WT mouse

tag: age

value: P60 (day after birth)


title: Con_PDX1-NKX6.1-pos_Rep1


title: Vehicle control 1

first: Christine

last: Becker

address: 1470 Madison Avenue

description: phHSC-AZ-Vehicle-72h---Ex1_CGGACAAC_AATCCGGA_HLVTNBGX9_R1_001

data_processing:
RNASeq raw data were processed using FAST QC software (http://www.bioinformatics.babraham.ac.uk/projects/fastqc/).
Data were aligned with STAR aligner via a computational pipeline implemented in NextFlow (Di Tommaso et al.,  2017)
After alignment and summarization with featureCounts, data was normalized.
Supplementary_files_format_and_content: Tab delimited text files

source_name: primary human hepatic stellate cells

growth_protocol: 200,000 Primary human hepatic stellate cells (phHSCs) per well were plated in 6-well plates.

extract_protocol:
Cells were harvested and total RNA was extracted using RNeasy Mini Kit. The purity of RNA (RNA integrity number or RIN) was assessed using 2100 Bioanalyzer instrument (Agilent, CA).
To prepare sequencing libraries from mRNA we used TruSeq Stranded mRNA library prep kit (Illumina, CA).   Sequencing was performed on NextSeq Hi (Illumnina, CA) sequencing instrument with setting at following output: R1=100; index=8; R2=100.

treatment_protocol: After overnight serum starvation the cells were treated with either vehicle or 30 nM of AZD3355 in DMEM with 0.1% BSA for 72 hours.

tag: treatment

value: Vehicle control

tag: timepoint

tag: cell type

value: primary human hepatic stellate cells


title: Vehicle control 2

description: phHSC-AZ-Vehicle-72h---Ex2_GCGCAAGC_CGGCGTGA_HLVTNBGX9_R1_001


title: condW_rep2_time6

city: Mannheim

first: Johanna

middle: Daniela

last: Schott

address: Franz-Volhard-Straße 6

department: Mannheim Institute for Innate Immunoscience

institute: Heidelberg University

data_processing:
Reads were mapped with STAR v2.5.3a (Dobin et al., Bioinformatics 2012) to a common reference of the human genome (hg38) and the ERCC92 RNA Spike-In Mix, providing the basic set of Gencode V27 as downloaded from the UCSC Genome Browser wgEncodeGencodeBasic27 table as transcript annotations, allowing up to 2 mismatches and chimeric read detection with a minimum of 10 nt per segment  (--chimSegmentMin 10).
Read counts were summarized at the gene level with the featureCounts function of the subread package v1.6.3 (Liao et al., Bioinformatics 2014), requiring that a read is completely contained within an exon to be counted.
For normalization, read counts were divided by the sum of all reads assigned to ERCC92 spike-in sequences in the corresponding sample.
Linear regression was performed on log-transformed read counts. Half-lives were calculated from the slope of the regression line with the equation: ln(2) / -slope. Only genes with a positive half-life and a coefficient of determination  R2 of >0.5 in all conditions were used for subsequent analyses.
Genome_build: hg38
Supplementary_files_format_and_content: csv file containing raw read counts per gene, expression values after normalization to spike-in RNA reads, half-lives in hours and the coefficient of determination (R2) of the linear regression used for determination of half-lives

source_name: HeLa RNF219 KO + RNF219-WT

growth_protocol: HeLa cells were cultured in Dulbecco's modified Eagle's medium (DMEM, Gibco) supplemented with 10% (v/v) fetal bovine serum (FBS, Sigma), 2 mM L-Glutamine, 100 U/ml penicillin and 0.1 mg/ml streptomycin (all PAN Biotech) at 37°C in a humidified atmosphere with 5% CO2. Every two to three days, cells were carefully detached by scraping in warm 1x PBS and diluted 1:10. On the day before experiments, cells were seeded at a density of 0.26 x 105 cells/cm2.

extract_protocol:
RNA was extracted with the GeneMatrix universal RNA purification kit (Roboklon). 2 µl of 1:100 diluted ERCC RNA Spike-In Mix (Thermo Scientific) was added per µg total RNA.
After depletion of rRNA using the Ribo-Zero Gold Kit (Illumina), sequencing libraries were prepared with the NEBNext Ultra II Directional RNA Library Prep Kit (NEB) and sequenced on a NextSeq550 system (Illumina) with 75 bases single end.

treatment_protocol: Transcription was inhibited by adding 5 µg/ml Actinomycin D (ApplicChem, A1489,0005) for 0, 2, 4 and 6 hours.

tag: cell line

value: HeLa

value: RNF219 KO + RNF219-WT

value: ActD, 6h


title: condM_rep2_time4

source_name: HeLa RNF219 KO + RNF219-mut

value: RNF219 KO + RNF219-mut

value: ActD, 4h


title: SAMPLE 23: wt3 30min

type: RNA

source_name: wt culture 3; 30 min after Zn treatment

tag: strain


title: Con_PP_Rep5

value: pancreatic progenitors


title: condW_rep2_time4


title: SAMPLE 20: wt3 2min

source_name: wt culture 3; 2 min after Zn treatment


title: SAMPLE 21: wt3 5min

source_name: wt culture 3; 5 min after Zn treatment


title: Con_PP_Rep4


title: condW_rep2_time2

value: ActD, 2h


title: SAMPLE 22: wt3 15min

source_name: wt culture 3; 15 min after Zn treatment

value: PAO1 WT Laboratory strain


title: Con_PP_Rep3


title: Con_PP_Rep2


title: Con_PE_Rep6

value: pancreatic endoderm


title: Con_PE_Rep5

value: embryonic stem cells, HUES8


title: Con_PE_Rep4


title: condKO_rep2_time0

source_name: HeLa RNF219 KO

value: RNF219 KO

value: untreated


title: SAMPLE 15: wt2 30min

source_name: wt culture 2; 30 min after Zn treatment

value: zinc induction


title: condKO_rep2_time4


title: SAMPLE 17: wt3 t0

source_name: wt culture 3; 0 s after Zn treatment


title: SAMPLE 18: wt3 0,5min

source_name: wt culture 3; 0.5 s after Zn treatment


title: condHeLa_rep2_time4

source_name: HeLa parental

value: parental


title: condKO_rep2_time6


title: SAMPLE 19: wt3 1min

source_name: wt culture 3; 1 min after Zn treatment


title: condW_rep2_time0


title: Con_PE_Rep3

value: wild type


title: Con_PE_Rep2


title: SAMPLE 16: wt2 60min

source_name: wt culture 2; 60 min after Zn treatment


title: SAMPLE 13: wt2 5min

source_name: wt culture 2; 5 min after Zn treatment


title: Con_PP_Rep1


title: condHeLa_rep2_time2


title: SAMPLE 12: wt2 2min

source_name: wt culture 2; 2 min after Zn treatment


title: Con_PE_Rep1


title: SAMPLE 11: wt2 1min

source_name: wt culture 2; 1 min after Zn treatment


title: Con_DE_Rep6

value: definitive endoderm


title: condKO_rep2_time2


title: condHeLa_rep2_time6


title: condM_rep1_time6


title: condM_rep1_time4


title: condHeLa_rep2_time0


title: Con_DE_Rep5


title: condM_rep1_time2


title: SAMPLE 14: wt2 15min

source_name: wt culture 2; 15 min after Zn treatment


title: SAMPLE 10: wt2 0,5min

source_name: wt culture 2; 0.5 s after Zn treatment


title: Con_DE_Rep4


title: Con_PDX1-NKX6.1-pos_Rep6

value: pancreatic progenitors, sorted PDX1+/NKX6.1+


title: SAMPLE 9: wt2 t0

source_name: wt culture 2; 0 s after Zn treatment


title: SAMPLE 8: wt1 60min

source_name: wt culture 1; 60 min after Zn treatment


title: SAMPLE 6: wt1 15min

source_name: wt culture 1; 15 min after Zn treatment


title: SAMPLE 5: wt1 5min

source_name: wt culture 1; 5 min after Zn treatment


title: Con_DE_Rep3


title: Con_DE_Rep2


title: condW_rep1_time4

source_name: HeLa RNF219 KO + RNF219-WT

value: HeLa


title: Con_DE_Rep1


title: Con_ESC_Rep6

value: undifferentiated


title: SAMPLE 7: wt1 30min

source_name: wt culture 1; 30 min after Zn treatment


title: Con_ESC_Rep5


title: condW_rep1_time6

value: RNF219 KO + RNF219-WT

value: ActD, 6h


title: condW_rep1_time2


title: condW_rep1_time0


title: condM_rep1_time0

source_name: HeLa RNF219 KO + RNF219-mut

value: RNF219 KO + RNF219-mut


title: SAMPLE 3: wt1 1min

source_name: wt culture 1; 1 min after Zn treatment


title: SAMPLE 4: wt1 2min

source_name: wt culture 1; 2 min after Zn treatment


title: condKO_rep1_time6

source_name: HeLa RNF219 KO


title: Con_ESC_Rep4


title: SAMPLE 1: wt1 t0

source_name: wt culture 1; 0 s after Zn treatment


title: condKO_rep1_time4

value: ActD, 4h


title: condKO_rep1_time2

value: ActD, 2h


title: Con_ESC_Rep1


title: Con_ESC_Rep3


title: KO input

city: Wuhan

first: Andi

last: Wang

address: Luojiashan Road

country: China

institute: Wuhan University

library_source: genomic

data_processing:
Cutadapt (version 1.16, http:// cutadapt.readthedocs.io/en/stable/guide.html) was used for adaptor sequence remove with the parameters -u 0 -u -30 -U 0 -U -30 -m 30
Cleaned reads were aligned to the mouse reference genome (mm10) using BWA (version 0.7.15, http://bio-bwa.sourceforge.net) with the default settings
Peaks calling was finished by MACS2 (version 2.1.1, https://github.com/taoliu/MACS) with the parameters --nomodel --keep-dup all -p 1E-10 --broad --broad-cut- off 1E-10 --extsize 147
Genome_build: mm10
Supplementary_files_format_and_content: bigwig

molecule: genomic DNA

source_name: neurospheres

extract_protocol:
For each experiment, single-cell suspensions from E12.5 neurospheres (passage 3) were collected. Cells were cross-linked with 1% formaldehyde for 10 min at room temperature and then quenched with 0.125 M glycine for 5 min. Cross-linked samples were then rinsed in PBS twice, and harvested in ice-cold IP buffer [100 mM NaCl, 50 mM tris-HCl (pH 8.1), 5 mM EDTA (pH 8.0), 0.02% NaN3, 0.5% SDS, 1× protease inhibitor cocktail, and 1 mM phenylmethylsulfonyl fluoride], followed by sonication in a Bioruptor Pico (Diagenode) at a setting of “30 s on/30 s off, 30 cycles” at 4°C. Twenty microliters of aliquot was taken for checking the efficiency of sonication. Thirty microliters of lysate (3%) was kept to quantify DNA before immunoprecipitation (input). Immunoprecipitation was performed overnight at 4°C on a rotating wheel with sheared chromatin and indicated antibodies: 3 μg of rabbit anti-SMARCA4/BRG1 antibody (Proteintech, 21634-1-AP), and 3 μg of control IgG antibody (ABclonal, AC005). The next day, immunocomplexes were incubated with 50 μl of Protein G Sepharose beads for 4 hours at 4°C, followed by washing three times with wash buffer I [20 mM tris-HCl (pH 8.0), 150 mM NaCl, 2 mM EDTA, 1% Triton X-100, and 0.1% SDS] and once with wash buffer II [20 mM tris-HCl (pH 8.0), 500 mM NaCl, 2 mM EDTA, 1% Triton X-100, and 0.1% SDS]. Protein-DNA complexes were decross-linked in 120 μl of elution buffer (1% SDS and 0.1 M sodium bicarbonate) by shaking at 65°C for 3 hours. The elution was incubated overnight at 65°C with 2 μl of proteinase K (20 mg/ml) (Sangon Biotech) and 2 μl of RNase A (10 mg/ml) (TAKARA). DNA extraction, precipitation, and resuspension were performed using a DNA purification kit (QIAGEN).
ChIP-seq libraries were constructed by StepWise DNA Lib Prep Kit for Illumina (Abclonal RK20202). For each sample, 40 μl purified ChIP DNA (about 300 pg) was end-repaired for dA tailing, followed by adaptor ligation with Full DNA Adapter Kit for Illumina Set_A (Abclonal RK20282). Each adaptor was marked with an index of 6 bp, which can be recognized after mixing different samples together. Adaptor-ligated ChIP DNA was purified by AMPure XP beads (1:1) and then used as template for 16 cycles of PCR amplification. Amplified ChIP DNA was purified again using AMPure XP beads (1:1) in 22 μl low-EDTA TE buffer. For multiplex sequencing, libraries with different index were mixed together with equal molar quantities by considering appropriate sequencing depth (20 million reads per library). Libraries were sequenced by Illumina Hi-seq X Ten platform with pair-end reads of 150 bp.

value: cortex

tag: developmental stage

value: Embryo 12.5

value: neurospheres

value: LncBAR-KO

tag: chip antibody

value: none (input)


title: condHeLa_rep1_time6

source_name: HeLa parental


title: condHeLa_rep1_time4


title: condHeLa_rep1_time2


title: WT rep2

value: WT


title: condKO_rep1_time0

value: RNF219 KO

value: untreated


title: HPAECs, Normoxia-2

city: Pittsburgh

first: Adam

last: Handen

state: Pennsylvania

address: 200 Lothrop Street

department: Vascular Medicine Institute

institute: University of Pittsburgh

description: HPAECs under normoxia and treated with vehicle

hyb_protocol: RNA samples were probed using the Clariom S Human Array.

scan_protocol: GeneChips were scanned using the GeneChip Scanner 3000 7G System.

data_processing: The gene expression was normalized with Robust Multiarray Analysis (RMA). The differential expression of the genes was determined using Affymetrix transcriptome analysis console (TAC) software.

label: biotin

source_name: primary pulmonary artery endothelial cells

label_protocol: RNA samples were labeled using the Affymetrix WTPLus kit according to manufacturer’s guidelines.

growth_protocol: Pulmonary arterial endothelial cells  (PAECs) plated in 6-well cell culture plates at 1X 10^5 cells per well, allowed to grow for 24 h, and the media were then changed to standard media and then the cells were placed into the hypoxia chamber.

extract_protocol: RNeasy kit (Qiagen) using extraction of total RNA was performed according to the manufacturer's instructions.

treatment_protocol: PAECs were treated with with vehicle control (DMSO, Sigma-41639), BRD2889 (1uM) (Broad Institute), as indicated. After exposure to hypoxia for 24 h, the cells were harvested for assay.

tag: stress

value: normoxia

value: control


title: condHeLa_rep1_time0

value: HeLa

value: parental


title: KO rep2


title: Con_ESC_Rep2

value: embryonic stem cells, HUES8

value: wild type

value: undifferentiated


title: KO rep1


title: WT input


title: Pcf [RM2]

city: Beijing

first: Lin

last: Sun

address: Xinjiekouwai Street

institute: Beijing Normal University

description: pcf11 duplication male

data_processing:
Basecalls performed using CASAVA.
Transcripts were quantified using Salmon (version 1.4.0).
Genome_build: Drosophila_melanogaster.BDGP6.28.dna.toplevel.fa
Supplementary_files_format_and_content: Matrix table of average expression and ratio

organism: Drosophila melanogaster

source_name: Pcf

growth_protocol: All the stocks are incubated at 25 degree

extract_protocol:
Total RNA was isolated using TRIZOL Reagent
RNA libraries were prepared for sequencing using standard Illumina protocols

tag: biological replicates

value: pcf11 duplication

tag: Sex

value: male

value: third-instar larvae


title: H3K4me3_input_24h

city: Ahmedabad

first: Noopur

last: Thakur

state: Gujarat

address: Navrangpura

department: Biological and Life Sciences

country: India

institute: Ahmedabad University

data_processing:
FastQC (v 0.11.9) was performed on Raw FastQ files fo Quality Check
Sequence read were trimmed where necessary to only retain high quality sequence. In addition, the low-quality sequence reads were excluded from the analysis. The adapter trimming was performed using Trimmomatic (Ver-0.36)
FastQ files were aligned to hg19 Reference Genome
The paired-end reads were aligned to the Human reference genome (hg19) Feb. 2009 release downloaded from UCSC database (GRCh37/hg19). The chromosome fasta file was downloaded from the following website (http://hgdownload.soe.ucsc.edu/goldenPath/hg19/bigZips/chromFa.tar.gz). The chromosome fasta file was downloaded from the following website and the Alignment was performed using BWA MEM (Ver-0.7.12).
Model-based Analysis of ChIP-Seq (MACS 2.1.1.20160309) was used to call peaks from aligned sequences with the following parameters: q value cutoff = 0.05, mfold = 5-50, bandwidth = 300 and Paired-end mode = ON.
Genome_build: hg19
Supplementary_files_format_and_content: peak files in BED format

source_name: PC-3 Cells

growth_protocol: PC-3 Cells were routiney maintained and Subcultured in RPMI-1640 medium (Himedia, Cat# AT028) supplemeted with 10% FBS ( Gibco, Cat# 10270106) and 1% Antibiotic-Antimycotic (Himedia, Cat# A002A) in 5% CO2 atmosphere.

extract_protocol:
TGF-β1 stimulated cells were treated with Formaldehyde to a final concentration of 1% to crosslink Proteins to DNA for 10 minutes at room temperature. Glycine was added at a final concentration of 125mM to quench the formaldehyde. Cells were washed twice with Phosphate Buffered Saline, scrapped and centrifuged. The cell pelelt was resuspended in ChIP lysis buffer (50 mM HEPES-KOH pH7.5, 140 mM NaCl, 1 mM EDTA pH8, 1% Triton X-100, 0.1% Sodium Deoxycholate, 0.1% SDS and Protease Inhibitors). Cell were lysed and sonicated using Diagenode Bioruptor Pico under 30 seconds ON and 30 seconds OFF for 30 cycles condition to generate DNA-Protein Complex fragments. 50uL of chromatin was removed to serve as input sample and rest of the chromatin was immunoprecipitated using ChIP-grade H3K4me3 Antibody (Abcam, Cat# 8580). The immunoprecipated chromatin was purified using Protein G Sepharose beads and washed once in low salt wash buffer, once in high salt wash buffer and once in LiCl wash buffer.  DNA was eluted using elution buffer and treated with 5M NaCl to reverse cross-link followed by treatement with RNaseA (Invitrogen, Cat# 12091021) and Proteinase K (Sigma-Aldrich, Cat#P2308). DNA was purified using PCR Purification kit (Himedia, Cat# MB512) and was stored at -20℃ until further use.
Libraries for ChIP sequencing were prepared using NEBNext® Ultra II DNA Library preparation kit (Cat# E7645). In brief, ChIP DNA and the Input DNA were subjected to various enzymatic steps for repair the ends and tailing with dA-tail followed by ligation of adapter sequences. These adapter ligated fragments were then size selected using SPRI beads. Next, the size selected fragments were indexed during limited cycle PCR to generate final libraries for paired-end sequencing

treatment_protocol: TGF-β1 (Abcam, Cat# AB50036) was reconstituted in 10mM Citric Acid (pH 3.0). 105 PC-3 Cells were seeded into T-25 Flask and incubated at 37℃ in 5% CO2 atmosphere till 70% confluency was achieved. Cells were serum-starved for 24hrs. Serum-Starved cells were then stimulated with TGF-β (5ng/mL) for 6Hrs and 24Hrs. Untreated cells were used as Control.

tag: disease

value: grade IV, adenocarcinoma

value: prostate; derived from metastatic site:bone

value: Treated with TGF-β1 for 24Hrs


title: H3K4me3_input_6h

value: Treated with TGF-β1 for 6Hrs


title: H3K4me3_24h

value: H3K4me3 (Abcam, ab8580)


title: SAMPLE 2: wt1 0,5min

source_name: wt culture 1; 0.5 s after Zn treatment

value: PAO1 WT Laboratory strain

value: zinc induction


title: WT rep1

value: cortex

value: Embryo 12.5

value: neurospheres

value: WT


title: HPAECs, Normoxia-3

value: primary pulmonary artery endothelial cells

value: normoxia

value: control


title: HPAECs, Hypoxia+BRD-2

description: HPAECs under hypoxia and treated with BRD-K34222889

value: hypoxia

value: BRD


title: HPAECs, Hypoxia-3

description: HPAECs under hypoxia and treated with vehicle.


title: H3K4me3_input_control

value: Control


title: H3K4me3_6h

value: grade IV, adenocarcinoma


title: HPAECs, Normoxia-1


title: Klf5-KO Lgr5+ ISC H3K27ac rep 1

city: Boston

first: Ramesh

last: Shivdasani

state: MA

address: 450 Brookline Ave. Dana 720D

department: Medical Oncology

institute: Dana Farber Cancer Institute

data_processing:
ChIP-seq alignment: Bowtie2
ChIP-seq differential region identification: diffReps; For visualization: merged bam files were used to create bigwigs and Ctrl and KO files were quantile-normalized using Haystack.
Genome_build: Mm10
Supplementary_files_format_and_content: ChIP-seq: bigwig files for visualizing aligned sequence tags.

source_name: Klf5-KO mouse intestinal stem cells

extract_protocol:
Proximal 1/3 of small intestine were resected, and crypts were isolated by villi scraping and 5mM EDTA incubation. Cells were dissociated with 4x TrpLE in DMEM for 45 minutes, and Lgr5-EGFPhi cells were sorted by flow cytometry. For ChIP-seq, whole cell extracts from sorted Lgr5-EGFPhi cells fixed with 1% formaldehyde were sonicated and incubated overnight with H3K27ac antibody (#39133, Active Motif) for immunoprecipitation.
ThruPLEX DNA-seq Kit (Rubicon Genomics) was used to prepare libraries.

treatment_protocol: Mice were injected with tamoxifen (1mg in 0.1 mL corn oil) for 5 consecutive days to activate Cre recombinase for initating lineage-tracing, and to delete Klf5 in Klf5-KO mice

value: Adult small intestinal stem cell; proximal 1/3 of intestine

tag: antibody

value: H3K27ac (#39133, Active Motif)


title: Lgr5+ ISC Input


title: Pcf [RM1]

description: pcf11 duplication male

value: pcf11 duplication

value: male

value: third-instar larvae


title: Control Lgr5+ ISC H3K27ac rep 2

source_name: Control mouse intestinal stem cells


title: H3K4me3_control

value: prostate; derived from metastatic site:bone

value: H3K4me3 (Abcam, ab8580)


title: Control Lgr5+ ISC H3K27ac rep 1


title: Klf5-KO Lgr5+ ISC H3K27ac rep 2


title: PRDM9 Amplicon Sequencing Pacbio CCS

status: Public on Oct 26 2021

city: Bethesda

first: Kevin

last: Brick

state: MD

address: 5/205 Memorial Drive

department: GBB

institute: NIDDK

description:
PRDM9 amplicon sequencing using Pacbio CCS sequencing
File_S1_PRDM9_genotypes.txt

data_processing:
Basecalling was performed using the Pacbio ccs tool (bioconda channel pbccs-4.2.0.0) and default parameters. The sequences of all possible barcode combinations from our dual barcoding approach were appended to a barcodes FASTA file. The reverse complement of each barcode was also included. Demultiplexing was performed using the Pacbio lima tool (bioconda channel pblima-1.11.0) and the following command line arguments ( --ccs --guess 45 --peek 10000 --guess-min-count 5 --different --score-full-pass ). Only reads flanked by a barcode on one side and its reverse complement on the other were retained.     ** NOTE : FASTQ files have been artificially created from the FASTA output of the basecalling and demultiplexing pipeline. The FASTQ quality scores are meaningless placeholder text and should not be used **
PRDM9 alleles were genotyped from basecalled FASTA files using custom scripts (see Alleva et al. 2021; see also https://github.com/kevbrick/genotype_prdm9_LR)
To generate per-individual FASTA files from the non-demultiplexed pool FASTQ, use the following code at the teminal:     zcat pacbioCCS.fastq.gz |perl -lane 'if ($_ =~ /^@((\S+?)\|(\S+?)\|.+)/){$id = $1; $name="$2.$3.fasta"; open OUT, ">>", $name ; print OUT ">$id"; next}; if ($name){print OUT $_; $name=""; close OUT}'
Genome_build: hg38
Supplementary_files_format_and_content: File_S1_PRDM9_genotypes.txt contains PRDM9 genotyping information for all individuals

source_name: individuals from the 1000 genomes project

extract_protocol:
LongAmp Taq 2X Master Mix (M0287) from New England Biolabs Inc. was used for PCR amplification. Post-amplification, samples were individually tested for successful amplification and low presence of polymerase slippage (presence of DNA laddering/smearing) by running on agarose gel electrophoresis. Samples were re-amplified if there was extensive DNA laddering/smearing by visualization. Based on the Genome Reference Consortium Human Build 38 (PRDM9 allele with 13 ZFs): the final amplified product was 1,899bp which contained the 1,092bp C2H2 ZF array, 670bp of upstream flanking sequence, and 137bp of downstream flanking sequence to the PRDM9 ZF array. Of note, the total length of the final amplified product varied based on the number of ZFs present in the PRDM9 allele. Samples were then pooled and prepared for multiplexing. We performed dual-barcoding in order to sequence all 758 multiplexed individuals. The first round of barcoding was done by adding unique DNA barcode sequences to the 5’ end of the PCR primers, totaling eight primer pairs. After amplification and the addition of the first barcode, samples were pooled in groups of eight (each sample tagged with a separate barcode sequence) and subjected to a second round of barcoding. The second round of multiplexing was performed using the PCR Barcoding Expansion 1-96 kit (EXP-PBC096) from Oxford Nanopore Technologies (ONT), Inc. following the protocol detailed on their website (https://nanoporetech.com - PCR barcoding (96) amplicons).
Sequencing libraries for Pacbio were prepared using the SMRTbell Express Template Prep Kit 2.0 (Pacific Biosciences, CA) and sequenced using the PacBio Sequel II System to generate Circular Consensus Sequencing (CCS) PacBio reads. Sequencing was performed with a 0.5 hour pre-extension and 10 hour recording time and a second sequencing run was performed with a 2 hour pre-extension and 30 hour recording time.

tag: individuals

value: mixed individuals (see File_S1_PRDM9_genotypes.txt)

tag: sequencing technique

value: Pacbio Circular Consensus Sequencing

tag: sequencing instrument


title: ChIP-CC556_WT_H3K36me3

city: Montréal

first: Claudia

middle: L

last: Kleinman

state: Québec

address: 3999 Côte Ste-Catherine Road

department: Human Genetics

country: Canada

institute: Lady Davis Institute for Medical Research

data_processing:
For smRNA samples: smRNA sequencing reads were trimmed using Trimmomatic (v0.32), removing adaptor, other Illumina-specific sequences and low-quality bases at the end of each read, using a 4bp sliding window to trim where average window quality fell below 30 (phred33 < 30). Trimmed reads with less than 15 bases were discarded. The resulting clean set of reads were then aligned to the drosophila reference genome (dm6) using novoalign (v3.02.12) with a score difference of 0 for calling multiple mapping (-R 0), reporting a maximum of 100 alignments per read (-r All 100), setting miRNA mode (-m 100) and using -l 14 and -t 10,5.5 as alignment scoring parameters.
For RNA samples: RNA sequencing reads were trimmed using Trimmomatic (v0.32), removing adaptor and other Illumina-specific sequences as well as the first four bases from the start of each read, and low-quality bases at the end of each read, using a 4bp sliding window to trim where average window quality fell below 30 (phred33 < 30). Trimmed reads with less than 30 bases were discarded. The resulting clean set of reads were then aligned to the drosophila reference genomes (dm6) using STAR (v2.3.0e) with default parameters. Reads mapping to more than 10 locations in the genome (MAPQ < 1) were discarded.
for ChIP samples: For ChIP-seq data, reads were trimmed of TruSeq adaptor sequences using trimmomatic. Trimmed reads were then aligned to dm6 using BWA-MEM, PCR duplicates were soft-clipped using Picard. BigWig files were generated using Homer, and consist of uniquely aligned reads without PCR duplicates.
Genome_build: dm6
Supplementary_files_format_and_content: bigwig files were generated by bedtools (v2.27.1) genomeCoverageBed, using the bam generated by STAR (RNA files) or Novoalign (smRNA files) and a normalization factor equal to the number of mapped reads divided by 100 million. bedGraph files were generated by HOMER, using the bam generated by bwa-mem.

source_name: 3rd instar larvae eye discs

extract_protocol:
RNA was extracted from Drosophila 3rd instar larvae eye discs using a Qiagen kit followed by DNAse treatment. For smRNA-seq RNA was extracted from Drosophila 3rd instar eye discs using Qiagen kit with Trizol, spike-in controls added at the beginning of the protocol, and DNAse treatment was added at the end. For crosslinked ChIP, cells were crosslinked (1% formaldehyde, 10mins) and sonicated. Protein:DNA complexes were immunoprecipitated with specific antibodies pre-bound to protein A or goat anti-mouse IgG magnetic beads. Eluted DNA underwent crosslink reversal, and were purified using Qiagen PCR purification columns. For RNA extraction, 1 million TEX cells were pelleted, washed with PBS and resuspended in 350ul RLT buffer (Qiagen-RNAeasy mini kit) with beta-mercaptoethanol. The suspension was then stored at -80oC until frozen. Sample was thawed and RNA extraction was performed using Qiagen's RNAeasy mini kit as per the manufacturer's instructions.
total RNA samples submitted to Genome Quebec facility for quality control assessment and library preparation. Illumina HiSeq 4000 with miRNA size select has been used for smRNAseq and Illumina HiSeq 4000 using mRNA stranded library has been used for RNA-seq. The eluted ChIP DNA undewent adaptor ligation based library construction for single-end 50bp Illumina sequencing on HiSeq 4000. RNA-seq library preparation was performed with ribosomal RNA (rRNA) depletion according to instructions from the manufacturer (Epicentre) to achieve greater coverage of mRNA and other long non-coding transcripts, paired-end sequencing (100 bp) was performed on the Illumina HiSeq 2500 or 4000 platform.

value: 3rd instar larvae

tag: tissue

value: eye-antennal imaginal discs

tag: genotype

value: WT H3K36me3


title: RNA-K36M_mCherryRNAi_3

value: K36M mCherryRNAi


title: ChIP-CC546_K36M_H3K36me3

value: K36M H3K36me3


title: HPAECs, Hypoxia+BRD-1

value: primary pulmonary artery endothelial cells

value: hypoxia

tag: treatment

value: BRD


title: HPAECs, Hypoxia-2

value: control


title: RNA-K36M_mCherryRNAi_2


title: RNA-K36M_KrimpRNAi_35230_2

value: K36M KrimpRNAi_35230


title: HPAECs, Hypoxia+BRD-3


title: RNA-K36M_KrimpRNAi_35230_3


title: RNA-K36M_EzRNAi_3

value: K36M EzRNAi


title: Control  [CM1]

description: wild type male

source_name: Control

value: wild type

value: male


title: PRDM9 Amplicon Sequencing Nanopore

description:
PRDM9 amplicon sequencing using Oxford Nanopore sequencing
File_S1_PRDM9_genotypes.txt

data_processing:
To identify sequencing reads derived from each individual, we performed read demultiplexing using Guppy v3.1.5. This first involved base calling (with standard parameters), followed by two rounds of demultiplexing to identify the outer and inner barcodes. The first round of demultiplexing identified the outer barcode as follows:    guppy_barcoder --compress_fastq -i {guppy output}       -s demux       --arrangements_files barcode_arrs_pcr96.cfg       --min_score 50 --front_window_size 300      --rear_window_size 300       --trim_barcodes    The second round of demultiplexing was then performed on each of the files generated from the first round:    guppy_barcoder --compress_fastq -i {round 1 barcoding FAST5}       --arrangements_files custom_12bp.cfg      --min_score 70 --front_window_size 100       --rear_window_size 100       --trim_barcodes    We used the Oxford Nanopore development basecaller Bonito (v.0.2.3) for base calling as it is more accurate than Guppy, the production basecaller (Silvestre-Ryan and Holmes 2020). Specifically, we found that the Guppy base calling accuracy for CpG dinucleotides in particular contexts was insufficient to confidently infer PRDM9 genotypes using our methods (not shown). Reads from each individual were grouped and base called separately using Bonito (v.0.2.3) and default parameters.     ** NOTE : FASTQ files have been artificially created from the FASTA output of hte Bonito basecaller. The FASTQ quality scores are meaningless placeholder text and should not be used **
PRDM9 alleles were genotyped from basecalled FASTA files using custom scripts (see Alleva et al. 2021; see also https://github.com/kevbrick/genotype_prdm9_LR)
To generate per-individual FASTA files from the non-demultiplexed pool FASTQ, use the following code at the teminal:     zcat bonito1d.fastq.gz |perl -lane 'if ($_ =~ /^@((\S+?)\|(\S+?)\|(\S+?)\|.+)/){$id = $1; $name="$2.$3_$4.fasta"; open OUT, ">>", $name ; print OUT ">$id"; next}; if ($name){print OUT $_; $name=""; close OUT}'
Genome_build: hg38
Supplementary_files_format_and_content: File_S1_PRDM9_genotypes.txt contains PRDM9 genotyping information for all individuals

extract_protocol:
LongAmp Taq 2X Master Mix (M0287) from New England Biolabs Inc. was used for PCR amplification. Post-amplification, samples were individually tested for successful amplification and low presence of polymerase slippage (presence of DNA laddering/smearing) by running on agarose gel electrophoresis. Samples were re-amplified if there was extensive DNA laddering/smearing by visualization. Based on the Genome Reference Consortium Human Build 38 (PRDM9 allele with 13 ZFs): the final amplified product was 1,899bp which contained the 1,092bp C2H2 ZF array, 670bp of upstream flanking sequence, and 137bp of downstream flanking sequence to the PRDM9 ZF array. Of note, the total length of the final amplified product varied based on the number of ZFs present in the PRDM9 allele. Samples were then pooled and prepared for multiplexing. We performed dual-barcoding in order to sequence all 758 multiplexed individuals. The first round of barcoding was done by adding unique DNA barcode sequences to the 5’ end of the PCR primers, totaling eight primer pairs. After amplification and the addition of the first barcode, samples were pooled in groups of eight (each sample tagged with a separate barcode sequence) and subjected to a second round of barcoding. The second round of multiplexing was performed using the PCR Barcoding Expansion 1-96 kit (EXP-PBC096) from Oxford Nanopore Technologies (ONT), Inc. following the protocol detailed on their website (https://nanoporetech.com - PCR barcoding (96) amplicons).
Sequencing libraries for Nanopore were prepared using either the 1D (SQK-LSK308) or the 1D2 sequencing kit (SQK-LSK309) from Oxford Nanopore Technologies and were run on a MinION sequencer with R9.5.1 flow cells (FLO-MIN107).

value: Oxford Nanopore

value: Oxford Nanopore FLO-MIN107


title: RNA-K27M_mCherryRNAi_3

value: K27M mCherryRNAi


title: RNA-K27M_mCherryRNAi_1


title: RNA-K27M_KrimpRNAi_35230_2

value: K27M KrimpRNAi_35230


title: RNA-K27M_Ash1RNAi_36130_3

value: 3rd instar larvae

value: K27M Ash1RNAi_36130


title: HPAECs, Hypoxia-1


title: Control [CM2]

description: wild type male

value: third-instar larvae


title: RNA-K27M_Ash1RNAi_36130_1

value: eye-antennal imaginal discs


title: RNA-K27M_Ash1RNAi_108832_3


title: RNA-ey_mcherryRNAi_3

value: ey mcherryRNAi


title: RNA-K27M_Ash1RNAi_108832_1


title: RNA-K36M_KrimpRNAi_35230_1


title: RNA-K36M_mCherryRNAi_1

value: K36M mCherryRNAi


title: dme_H3K27M_Input

data_processing:
For smRNA samples: smRNA sequencing reads were trimmed using Trimmomatic (v0.32), removing adaptor, other Illumina-specific sequences and low-quality bases at the end of each read, using a 4bp sliding window to trim where average window quality fell below 30 (phred33 < 30). Trimmed reads with less than 15 bases were discarded. The resulting clean set of reads were then aligned to the drosophila reference genome (dm6) using novoalign (v3.02.12) with a score difference of 0 for calling multiple mapping (-R 0), reporting a maximum of 100 alignments per read (-r All 100), setting miRNA mode (-m 100) and using -l 14 and -t 10,5.5 as alignment scoring parameters.
For RNA samples: RNA sequencing reads were trimmed using Trimmomatic (v0.32), removing adaptor and other Illumina-specific sequences as well as the first four bases from the start of each read, and low-quality bases at the end of each read, using a 4bp sliding window to trim where average window quality fell below 30 (phred33 < 30). Trimmed reads with less than 30 bases were discarded. The resulting clean set of reads were then aligned to the drosophila reference genomes (dm6) using STAR (v2.3.0e) with default parameters. Reads mapping to more than 10 locations in the genome (MAPQ < 1) were discarded.
for ChIP samples: For ChIP-seq data, reads were trimmed of TruSeq adaptor sequences using trimmomatic. Trimmed reads were then aligned toBDGP5 using BWA-MEM, PCR duplicates were soft-clipped using Picard. BigWig files were generated using Homer, and consist of uniquely aligned reads without PCR duplicates.
Genome_build: BDGP5
Supplementary_files_format_and_content: bedGraph files were generated by HOMER, using the bam generated by bwa-mem.

value: 3rd instar larvae eye discs


title: RNA-ey_Ash1RNAi_2

value: ey Ash1RNAi


title: RNA-K27M_KrimpRNAi_35230_1


title: RNA-K27M_Ash1RNAi_36130_2


title: dme_H3K36M_H3K27me3


title: dme_H3WT_H3K27me3


title: dme_H3K36M_H3K36me2


title: RNA-K36M_EzRNAi_1


title: RNA-ey_Ash1RNAi_1


title: pombe_dus4_noR_II

city: Namur

first: Carlo

last: Yague-Sanz

address: 61 rue de Bruxelles

department: Medecine

country: Belgium

institute: Namur University

data_processing:
Library strategy: Rho-seq
Reads were quality- and adaptor-trimmed using trimmomatic 0.36 with options PE ILLUMINACLIP:TruSeq2-PE.fa:2:30:10 HEADCROP:1 SLIDINGWINDOW:4:15 MINLEN:20
The filtered reads were mapped on the spike-in sequence with bowtie2 (options --no-discordant --no-mixed --phred33 -p 4 --un-conc) then on the genome using also bowtie2 for the E.coli libraries or tophat2 for the S. pombe libraries (options--mate-std-dev 150 -N 2 --read-edit-dist 2 --min-intron-length 5 --max-intron-length 3000 --no-discordant --no-coverage-search -p 4 --library-type fr-firststrand).
Properly paired mapped reads were split by strand. Then bedtools (options genomecov -ibam -d -5) was used to calculate the number of RT termination events for each strand-specific position as the number of 5'end of R2 reads that map at this position. In parallel, the paired reads were computationally extended/merged into fragments that covered the genome from the left end of the left-most read to the right end of the right-most reads with the notable exeption that the fragments were not extended in identified introns. Those spliced fragments where then used to compute a fragment coverage reflecting the number of time a reverse transcriptase passed through a specific position. Then the ratio between the number of RT termination events and fragment coverage at each position (the D-ratio) was calculated.
After independent pre-filtering of the positions unlikely to have significant differences in D-ratio accross conditions (see manuscrit), the number of RT termination events out of the number of RT readthrough were modelised in generalized linear model of the binomial family (function glm in R) according to the effect of the treatment (R+ vs R-), the effect of the strain (wild-type vs delta4dus), and their interaction. The positions were the interaction between the wild-type and the delta4dus lead to a statistically significant (FDR<0.1) increase of the proportion of RT termination events were retained as putative dihydrouridilation sites.
Genome_build: ASM294v2.26 (pombe), ASM584v2.38 (coli), hg38 (human)
Supplementary_files_format_and_content: Rho-seq_supplementary.xlsx is a spreadsheet describing the dihydrouridilated sites (D-sites) uncovered in our study. The first three sheets describe the D-sites found respectively on the S. pombe, the E. coli and human transcriptome, while the third sheet contains a legend explaining the column names in the first three sheets.

organism: Schizosaccharomyces pombe

source_name: S. pombe cells

growth_protocol: 50 mL of S. pombe grown in YES at 32°C (OD595nm ~ 0.5) or E. coli grown in LB at 37°C (OD595nm ~ 0.6) or HCT116 human cells were harvested by centrifugation and washed in DEPC water.

extract_protocol:
The cell pellets were resuspended in 750μL of TES buffer (10mM Tris pH 7.5, 10mM EDTA pH 8, 0.5% SDS) and the total RNA was isolated by hot phenol extraction. Rhodamine labeling was performed as described in our manuscript. Briefly, 30μg of total RNA were resuspended in 1% sodium borohydride for 60min at 25°C. Mock samples (R-) were treated similarly but without sodium borohydride. The reduced RNA was then precipitated and the dried pellet was resuspended in 0.1M sodium formate pH 4. Rhodamine labeling was performed for 90min at 37°C in dark with 2.2mM of rhodamine chlorine. Labeled RNA was purified by phenol extraction.
A synthetic dihydrouridilated RNA spike-in was added to the samples (1:1000) before ribodepletion of 5 μg of the RNA samples. Zinc-mediated fragmentation was performed at 70°C for 15min with 90-125 ng of ribodepleted RNA. Simultaneous DNA digestion and RNA extremities dephosphorylation were performed at 37°C for 30min with 2U of TURBO DNase and 3U of FastAP Thermosensitive Alkaline Phosphatase. 3'-end ligation of the RNA adapter was performed for 90min at 25°C with 39U of T4 RNA ligase. After ligation, 10pmol of DNA primer were added, the sample was denatured for 2min at 70°C and the reverse transcription was performed with Superscript III. The excess of DNA primer was subsequently digested with 4min incubation with 2μL of ExoSAP-IT. The 3'-end ligation of the cDNA with DNA adapter was performed overnight at 25°C with 48U of T4 RNA ligase. Nucleic acid clean-ups were performed between each step by using MyOne SILANE magnetic beads. 13 cycles od PCR amplification was performed in a 50μL-reaction by mixing cDNA, 1μM of barcoded PCR primer, 1μM of universal PCR primer and 25μL of NEBNext High- Fidelity 2X PCR Master Mix (NEB M0541). Finally, two successive rounds of DNA polymer size selection were performed by mixing PCR products with 1.2 and 1.1 volume of AMPure XP beads. The DNA library was quantified, examined on a DNA microchip and sequenced on Illumina HiSeq 2500 or Novaseq.

tag: replicate

value: II

value: R-

tag: percentage of d in spike-in


title: RNA-K27M_Ash1RNAi_108832_2


title: RNA-K36M_EzRNAi_2

value: K36M EzRNAi


title: RNA-K27M_mCherryRNAi_2


title: RNA-K27M_KrimpRNAi_35230_3


title: pombe_dus3_noR_II


title: RNA-ey_mcherryRNAi_1


title: pombe_dus2_noR_II


title: dme_H3K36M_Input


title: dme_H3WT_H3K36me2


title: RNA-ey_EzRNAi_1

value: ey EzRNAi


title: dm_yw_H3K36me2

value: yw


title: pombe_WT_noR_II

value: WT


title: dme_H3K27M_H3K36me2


title: RNA-ey_mcherryRNAi_2


title: RNA-ey_EzRNAi_2


title: pombe_dus4_noR_I

value: I


title: pombe_dus4_R_I

value: R+


title: RNA-ey_Ash1RNAi_3


title: pombe_dus3_R_I


title: pombe_dus2_noR_I


title: dme_yw_H3K27me3


title: pombe_dus2_R_I


title: RNA-ey_EzRNAi_3


title: pombe_dus2_R_II


title: pombe_dus1_R_I


title: pombe_WT_noR_I


title: pombe_dus1_R_II


title: dme_yw_Input


title: dme_H3K27M_H3K27me3


title: pombe_dus3_R_II


title: pombe_WT_R_I


title: pombe_dus4_R_II


title: dme_H3WT_Input


title: pombe_dus1_noR_I


title: pombe_WT_R_II


title: pombe_dus3_noR_I


title: pombe_dus1_noR_II


title: FosKO_stim120.1

city: London

first: Steve

last: Ley

address: Pond Street

department: Institute of Immunity & Inflammation

country: United Kingdom

institute: University College London

data_processing:
The RSEM package (version 1.2.31) (Li and Dewey, 2011) was used in conjunction with the STAR alignment algorithm (version 2.5.2a) (Dobin et al., 2013) for the mapping and subsequent gene-level counting of the sequenced reads with respect to Ensembl mouse GRCm38 release 89  transcriptome
Normalisation of raw count data and differential expression analysis was performed with the DESeq2 package (version 1.16.1)  within the R programming environment (version 3.4.1)
Genome_build: GRCm38 release 89
Supplementary_files_format_and_content: VST log2 normalised expression values. Rows = genes; Columns = samples.

growth_protocol: Bone marrow cells from both femurs were cultured on non-adherent dishes for 7 days in RPMI 1640 supplemented with 20% L-cell conditioned medium, 10% FBS, 1 mM sodium pyruvate, 50 µM 2-Mercaptoethanol, 10mM HEPES, L-glutamine and pencillin/streptomycin. Cells were fed on day 4.

extract_protocol: RNA was extracted using the Rneasy kit (Qiagen) according to the manufacturers protocol. RNA was eluted in 50 µl Rnase-free water.

treatment_protocol: On day 7, cells were seeded in 6 well plates at 0.5 x 10^6 cells/ml in 2ml overnight in RPMI 1640 supplemented with 1% FBS, 1 mM sodium pyruvate, 50 µM 2-Mercaptoethanol, 10mM HEPES, L-glutamine and pencillin/streptomycin. Cells were then stimulated with 100 ng/ml LPS for the indicated times or left unstimulated

value: LPS (100ng/ml) for 2.0h

tag: molecule

value: total RNA

value: bone marrow-derived macrophages


title: FosKO_stim30.2

tag: strain

value: LPS (100ng/ml) for 0.5h


title: FosKO_stim30.3


title: FosKO_stim120.2


title: LysM_stim120.3


title: FosKO_stim60.2

value: LPS (100ng/ml) for 1.0h


title: LysM_stim120.2


title: FosKO_unstim.3

value: untreated


title: LysM_stim120.1


title: FosKO_stim120.3


title: FosKO_stim60.3


title: LysM_stim60.3


title: FosKO_unstim.1


title: FosKO_unstim.2


title: FosKO_stim30.1


title: FosKO_stim60.1


title: LysM_stim60.1


title: CLAP-seq_INPUT_dIDR_B [CLAP-seq]

city: Los Angeles

first: Kathrin

last: Plath

state: CA

address: 615 Charles E Young Dr S

institute: University of California, Los Angeles

description:
CLAP-seq of female ESCs with halo-tagged delta IDR-SPEN and tetO Xist on Bl6 X chromosome.
Mus musculus x Mus castaneous

data_processing:
Library strategy: CLAP-seq
[mRNA-seq]
Bulk RNA-seq data was aligned to the mm10 genome with STAR (v2.7.1a), filtered with samtools (v1.7) and allelic ratios were called using GATK (v4.1.4.1) ASEReadCounter.
Allelic reads were summed across genes. Genes with fewer than 10 allelic reads were filtered out from each sample. Allelic ratios between 0h and 24h dox were compared with a one-sided welch t-test (p<0.05), where each replicate is a sample.
[10X 3'GEX]
Cellranger (v5.0.1) was used to align and process the reads to the mm10 genome, Seurat (v3.9.9.9024) in R (v3.6.1) to filter out low quality cells, and vartrix (v1.1.14) to generate allelic ratios for each SNP in each cell.
Allelic reads were summarized across genes. Lowly expressed genes were filtered out, by requiring a minimum of 10 cells in each sample to have at least one allelic read expressed, and a minimum of 3 allelic reads per gene in each sample. Xist is induced by dox from the Bl6 allele, all cells were assumed to inactivate the Bl6 X chromosome. Allelic ratios between 0h and 24h dox for each mutant were compared using a one-sided welch t-test (p<0.05), where each cell is a sample.
[CLAP-seq]
CLAP-seq of full length, dIDR and dRRM SPEN was aligned to the mm10 genome with STAR (v2.7.1a), filtered with samtools (v1.7), duplicate reads were removed with GATK (v4.1.4.1) MarkDuplicates. RPKM was calculated for each sample in 100bp bins smoothed across 300bp with deeptools (v3.5.0), and enrichment was calculated by dividing SPEN pulldown RPKM over input RPKM in each bin.
Genome_build: mm10 (GRCm38)
Supplementary_files_format_and_content: SPEN_WT_dIDR_rescue_featurecounts.txt: featureCounts file.
Supplementary_files_format_and_content: spen_WT_KO_dIDR_rescue_bulk_ttest.csv: t-test results of 24h vs 0h allelic ratios in 3 replicates of WT, dIDR, and dSPOC.
Supplementary_files_format_and_content: features.tsv, barcodes.tsv, and matrix.mtx: Cellranger outputs.
Supplementary_files_format_and_content: spen_WT_dIDR_dSPOC_10x_ttest.csv: t-test results of 24h vs 0h allelic ratios in WT, dIDR, and dSPOC single cells.
Supplementary_files_format_and_content: DeltaB_WT_D2_ttest.csv: t-test results of deltaB vs WT allelic ratios in 129-Xist expressing single cells at day 2 of differentiation.
Supplementary_files_format_and_content: DeltaB_WT_D4_ttest.csv: t-test results of deltaB vs WT allelic ratios in 129-Xist expressing single cells at day 4 of differentiation.
Supplementary_files_format_and_content: dIDR_A.rpkm.Xist.bedgraph: RPKM of dIDR-SPEN binding to Xist RNA in 100bp bins smoothed across 300bp.
Supplementary_files_format_and_content: dIDR_B.rpkm.Xist.bedgraph: RPKM of dIDR-SPEN binding to Xist RNA in 100bp bins smoothed across 300bp.
Supplementary_files_format_and_content: dRRM_B.rpkm.Xist.bedgraph: RPKM of dRRM-SPEN binding to Xist RNA in 100bp bins smoothed across 300bp.
Supplementary_files_format_and_content: FL_A.rpkm.Xist.bedgraph: RPKM of FL-SPEN binding to Xist RNA in 100bp bins smoothed across 300bp.
Supplementary_files_format_and_content: FL_B.rpkm.Xist.bedgraph: RPKM of FL-SPEN binding to Xist RNA in 100bp bins smoothed across 300bp.
Supplementary_files_format_and_content: INPUT_dIDR_A.rpkm.Xist.bedgraph: RPKM of dIDR-SPEN input over Xist RNA in 100bp bins smoothed across 300bp.
Supplementary_files_format_and_content: INPUT_dIDR_B.rpkm.Xist.bedgraph: RPKM of dIDR-SPEN input over Xist RNA in 100bp bins smoothed across 300bp.
Supplementary_files_format_and_content: INPUT_dRRM_B.rpkm.Xist.bedgraph: RPKM of dRRM-SPEN input over Xist RNA in 100bp bins smoothed across 300bp.
Supplementary_files_format_and_content: INPUT_FL_B.rpkm.Xist.bedgraph: RPKM of FL-SPEN input over Xist RNA in 100bp bins smoothed across 300bp.
Supplementary_files_format_and_content: INPUT_FL_A.rpkm.Xist.bedgraph: RPKM of FL-SPEN input over Xist RNA in 100bp bins smoothed across 300bp.

organism: Mus musculus musculus x Mus musculus castaneus

source_name: ESC

growth_protocol: ESCs were grown in 2i + LIF medium.

extract_protocol:
Cells were lysed in iCLIP lysis buffer (50 mM Hepes, pH 7.4, 100 mM NaCl, 1% NP-40, 0.1% SDS, 0.5% Sodium Deoxycholate) supplemented with 1X Protease Inhibitor Cocktail (Promega), 200 U of Murine RNase Inhibitor (New England Biolabs), 20 U Turbo DNase (Ambion), and 1X Manganese/Calcium Mix (0.5mM CaCl2, 2.5 mM MnCl2).
RNA was dephosphorylated (Fast AP) and cyclic phosphates removed (T4 PNK). The RNA was then ligated to an RNA adapter containing a RT primer binding site. The ligated RNA was reverse transcribed (RT) into cDNA, the RNA was degraded using NaOH, and a second adapter was ligated to the single stranded cDNA. The DNA was amplified and Illumina sequencing adaptors were added by PCR using primers that are complementary to the 3’ and 5’ adapters.

value: female

tag: cell type

value: ESC (EpiLC)

tag: cell line

value: Stable expression of halo-tagged delta IDR SPEN

tag: xist

value: tetO-Xist

tag: differentiation

value: 4 day differentiation in EpiLC media

value: last 24h Dox


title: CLAP-seq_INPUT_dRRM_B [CLAP-seq]

description:
CLAP-seq of female ESCs with halo-tagged delta RRM-SPEN and tetO Xist on Bl6 X chromosome.
Mus musculus x Mus castaneous

tag: Sex

value: Stable expression of halo-tagged delta RRM 1-4 SPEN


title: CLAP-seq_INPUT_FL_B [CLAP-seq]

description:
CLAP-seq of female ESCs with halo-tagged full length-SPEN and tetO Xist on Bl6 X chromosome.
Mus musculus x Mus castaneous

value: Stable expression of halo-tagged full length SPEN


title: LysM_stim30.2


title: CLAP-seq_dIDR_B [CLAP-seq]


title: CLAP-seq_INPUT_FL_A [CLAP-seq]


title: LysM_unstim.3


title: LysM_stim60.2


title: DeltaB_D4 [single-cell RNA-seq]

description:
Single-cell RNA-seq of female ESCs with heterozygous B-repeat deletion on the 129S4/SvJae Xist allele at day 4 of EpiLC differentiation.
Mus musculus x Mus castaneous
10X 3'GEX
processed data file: DeltaB_WT_D4_ttest.csv

data_processing:
[mRNA-seq]
Bulk RNA-seq data was aligned to the mm10 genome with STAR (v2.7.1a), filtered with samtools (v1.7) and allelic ratios were called using GATK (v4.1.4.1) ASEReadCounter.
Allelic reads were summed across genes. Genes with fewer than 10 allelic reads were filtered out from each sample. Allelic ratios between 0h and 24h dox were compared with a one-sided welch t-test (p<0.05), where each replicate is a sample.
[10X 3'GEX]
Cellranger (v5.0.1) was used to align and process the reads to the mm10 genome, Seurat (v3.9.9.9024) in R (v3.6.1) to filter out low quality cells, and vartrix (v1.1.14) to generate allelic ratios for each SNP in each cell.
Allelic reads were summarized across genes. Lowly expressed genes were filtered out, by requiring a minimum of 10 cells in each sample to have at least one allelic read expressed, and a minimum of 3 allelic reads per gene in each sample. Xist is induced by dox from the Bl6 allele, all cells were assumed to inactivate the Bl6 X chromosome. Allelic ratios between 0h and 24h dox for each mutant were compared using a one-sided welch t-test (p<0.05), where each cell is a sample.
[CLAP-seq]
CLAP-seq of full length, dIDR and dRRM SPEN was aligned to the mm10 genome with STAR (v2.7.1a), filtered with samtools (v1.7), duplicate reads were removed with GATK (v4.1.4.1) MarkDuplicates. RPKM was calculated for each sample in 100bp bins smoothed across 300bp with deeptools (v3.5.0), and enrichment was calculated by dividing SPEN pulldown RPKM over input RPKM in each bin.
Genome_build: mm10 (GRCm38)
Supplementary_files_format_and_content: SPEN_WT_dIDR_rescue_featurecounts.txt: featureCounts file.
Supplementary_files_format_and_content: spen_WT_KO_dIDR_rescue_bulk_ttest.csv: t-test results of 24h vs 0h allelic ratios in 3 replicates of WT, dIDR, and dSPOC.
Supplementary_files_format_and_content: features.tsv, barcodes.tsv, and matrix.mtx: Cellranger outputs.
Supplementary_files_format_and_content: spen_WT_dIDR_dSPOC_10x_ttest.csv: t-test results of 24h vs 0h allelic ratios in WT, dIDR, and dSPOC single cells.
Supplementary_files_format_and_content: DeltaB_WT_D2_ttest.csv: t-test results of deltaB vs WT allelic ratios in 129-Xist expressing single cells at day 2 of differentiation.
Supplementary_files_format_and_content: DeltaB_WT_D4_ttest.csv: t-test results of deltaB vs WT allelic ratios in 129-Xist expressing single cells at day 4 of differentiation.
Supplementary_files_format_and_content: dIDR_A.rpkm.Xist.bedgraph: RPKM of dIDR-SPEN binding to Xist RNA in 100bp bins smoothed across 300bp.
Supplementary_files_format_and_content: dIDR_B.rpkm.Xist.bedgraph: RPKM of dIDR-SPEN binding to Xist RNA in 100bp bins smoothed across 300bp.
Supplementary_files_format_and_content: dRRM_B.rpkm.Xist.bedgraph: RPKM of dRRM-SPEN binding to Xist RNA in 100bp bins smoothed across 300bp.
Supplementary_files_format_and_content: FL_A.rpkm.Xist.bedgraph: RPKM of FL-SPEN binding to Xist RNA in 100bp bins smoothed across 300bp.
Supplementary_files_format_and_content: FL_B.rpkm.Xist.bedgraph: RPKM of FL-SPEN binding to Xist RNA in 100bp bins smoothed across 300bp.
Supplementary_files_format_and_content: INPUT_dIDR_A.rpkm.Xist.bedgraph: RPKM of dIDR-SPEN input over Xist RNA in 100bp bins smoothed across 300bp.
Supplementary_files_format_and_content: INPUT_dIDR_B.rpkm.Xist.bedgraph: RPKM of dIDR-SPEN input over Xist RNA in 100bp bins smoothed across 300bp.
Supplementary_files_format_and_content: INPUT_dRRM_B.rpkm.Xist.bedgraph: RPKM of dRRM-SPEN input over Xist RNA in 100bp bins smoothed across 300bp.
Supplementary_files_format_and_content: INPUT_FL_B.rpkm.Xist.bedgraph: RPKM of FL-SPEN input over Xist RNA in 100bp bins smoothed across 300bp.
Supplementary_files_format_and_content: INPUT_FL_A.rpkm.Xist.bedgraph: RPKM of FL-SPEN input over Xist RNA in 100bp bins smoothed across 300bp.

extract_protocol:
Cells were dissociated with accutase for 5 minutes, washed 3 times with DPBS and resuspended in PBS containing 0.04% BSA. Cell concentration was adjusted between 800 to 1200 cells/μl and cell suspension was kept on ice before loading on the 10x Genomics Chromium instrument.
scRNA-seq libraries were generated using the Chromium single cell 3’ reagent kit V3.1.

value: 129S4/SvJae x Cast

value: Xist B repeat deleted on 129S4/SvJae allele

value: WT


title: CLA11-24h [single-cell RNA-seq]

description:
Single-cell RNA-seq of delta IDR-SPEN female ESCs with tetO-Xist allele on Bl6 X chromosome, with 24h dox-induced Xist expression.
Mus musculus x Mus castaneous
10X 3'GEX
processed data file: spen_WT_dIDR_dSPOC_10x_ttest.csv

value: ESC

value: KO of IDR domain of SPEN

value: 24h differentiation in MEF media

value: 24h Dox


title: CLAP-seq_dIDR_A [CLAP-seq]

description:
CLAP-seq of female ESCs with halo-tagged delta IDR-SPEN and tetO Xist allele on Bl6 X chromosome.
Mus musculus x Mus castaneous


title: CL87-0h [single-cell RNA-seq]

description:
Single-cell RNA-seq of delta SPOC-SPEN female ESCs with tetO Xist allele on Bl6 X chromosome.
Mus musculus x Mus castaneous
10X 3'GEX
processed data file: spen_WT_dIDR_dSPOC_10x_ttest.csv

value: KO of SPOC domain of SPEN


title: CLAP-seq_FL_A [CLAP-seq]


title: CLAP-seq_dRRM_B [CLAP-seq]


title: WT_D2 [single-cell RNA-seq]

description:
Single-cell RNA-seq of WT ESCs day 2 of EpiLC differentiation.
Mus musculus x Mus castaneous
10X 3'GEX
processed data file: DeltaB_WT_D2_ttest.csv

value: 2 day differentiation in EpiLC media


title: CLAP-seq_INPUT_dIDR_A [CLAP-seq]

value: female


title: LysM_unstim.1

value: untreated

value: total RNA

value: bone marrow-derived macrophages


title: DeltaB_D2 [single-cell RNA-seq]

description:
Single-cell RNA-seq of female ESCs with heterozygous B-repeat deletion on the 129S4/SvJae Xist allele at day 2 of EpiLC differentiation.
Mus musculus x Mus castaneous
10X 3'GEX
processed data file: DeltaB_WT_D2_ttest.csv

value: ESC (EpiLC)


title: LysM_stim30.1

value: LPS (100ng/ml) for 0.5h


title: CL53-24hDOX [single-cell RNA-seq]

description:
Single-cell RNA-seq of female ESCs with tetO Xist allele on Bl6 X chromosome, with 24h dox-induced Xist expression.
Mus musculus x Mus castaneous
10X 3'GEX
processed data file: spen_WT_dIDR_dSPOC_10x_ttest.csv

value: tetO-Xist


title: PPT

city: Yokohama

first: mami

last: kikegawa

state: Kanagawa

address: 601 Matanocho Totsuka-ku

department: Kampo Medicine

country: Japan

institute: Yokohama University of Pharmacy

data_processing:
Bcl2fastq (v2.17.1.14) software used for basecalling.
score is the ASCII value of the corresponding character minus 33. For example, the ASCII value of '@' is 64, and therefore the corresponding base quality score is 31 (64-33). Starting with Illumina GA Pipeline v1.8 (currently v1.9), base quality scores range from 0 to 41.
Reads Per Kilobase of exon per Megabase of library size (RPKM) were calculated using a protocol from Chepelev et al., Nucleic Acids Research, 2009. In short, exons from all isoforms of a gene were merged to create one meta-transcript. The number of reads falling in the exons of this meta-transcript were counted and normalized by the size of the meta-transcript and by the size of the library.
FPKM threshold for gene expression is set between 0.1-1
Genome_build: NOT PROVIDED
Supplementary_files_format_and_content: FPKM

source_name: mammary cell line

growth_protocol: MCF-7 cells were cultured in growth medium containing Dulbecco’s minimum essential medium (DMEM)/Ham’s F-12 with L-glutamine and sodium pyruvate without HEPES and phenol red (Cellgro by Mediatech Inc., Manassas, VA, USA) plus 10% fetal bovine serum (FBS) treated with charcoal/dextran (Hyclone by Thermo Scientific, South Logan, UT, USA), and 1% penicillin-streptomycin solution (100X) (P/S; FUJIFILM Wako Pure Chemical Co., Osaka, Japan) and maintained at 37°C and 5% CO2. MCF-7 cells were seeded in the growth medium.

extract_protocol:
Total RNA was isolated using the ReliaPrep™ RNA Miniprep Systems (Promega Co., Madison, WI, USA)
RNA libraries were prepared for sequencing using standard Illumina protocols

treatment_protocol: MCF-7 cells were seeded in 24-well plates with different concentrations of E2, PPD, and PPT for 48 h.

value: treated with PPT for 48 hours


title: CLAP-seq_FL_B [CLAP-seq]

value: Stable expression of halo-tagged full length SPEN

value: 4 day differentiation in EpiLC media

value: last 24h Dox


title: MCF7_H3K4me3_TET2_mCD_Rep2

city: Rennes

first: Gilles

last: Salbert

address: 263 Av. Gal. Leclerc

department: IGDR UMR6290

country: France

institute: University Rennes 1

data_processing:
All ChIP-seq and SCL-exo-seq sequencing reads were mapped to hg19 with using Bowtie (Langmead et al., Genome Biol. 2009). SAMtools (Li et al., Bioinformatics 2009) generated bam files which were processed with MACS (Zhang et al., Genome Biol. 2008) to generate wig files. Peak calling followed the procedure descibed in Sérandour et al (Nucleic Acids Res., 2012).
RNA-seq reads were mapped to hg19 with Bowtie (Langmead et al., Genome Biol. 2009) and transcripts were quantified with RSEM (Li and Dewey, Bioinformatics 2011). Differentially expressed genes were identified from RNA-seq data by the R package DESeq2 (Love et al., Genome Biol. 2014) after filtering of the raw data using the R package HTSFilter (Rau et al., Bioinformatics 2013).
SCL-exo-seq reads were treated as descibed (Sérandour et al., Bio-Protoc. 2018) using the python script available at https://mycore.core-cloud.net/index.php/s/4gyZ9dLTqgo86dt on SAM files.
Genome_build: hg19

source_name: TET2 mCD cells

growth_protocol: Cells were grown in high glucose and pyruvate containing DMEM (Gibco 41966) supplemented with 10% fetal calf serum (Eurobio S116365181H), non-essential amino acids (Gibco 11140035), Penicillin-Streptomycin (Gibco 15240) and Geneticin (Gibco 11811064) at 37°C and 5% CO2.

extract_protocol:
For ChIP-seq assays, cells were fixed in 1.5% formaldehyde (Sigma F8775) for 10 min at room temperature, the reaction was stopped by the addition of glycine (100 mM). Cells were lysed in lysis buffer (150 mM Tris-HCl pH 8.1, 10 mM EDTA, 0.5% Empigen BB, 1% SDS, protease inhibitor cocktail (Roche 5056489) and sonicated using a bioruptor (Diagenode, 15 min 30 sec ON /30 sec OFF). Sonicated chromatin was incubated at 4°C overnight with antibodies (ERa: mix of TE111.5D11 from Abcam and HC-20 from Santa Cruz Biotechnology, H3K4me3: 04-745 from Millipore, H3K27me3: 07-449 from Millipore) in IP buffer (2.8 ng/mL yeast tRNA, 20 mM Tris-HCl, 2 mM EDTA, 150 mM NaCl, 1% Triton X-100, proteases inhibitor cocktail). Complexes were recovered after 4h incubation with 45 mL protein A- and 15 mL protein G-conjugated sepharose beads slurry at 4°C. Beads were washed in washing buffers WB1 (20 mM Tris-HCl pH 8, 2 mM EDTA, 150 mM NaCl, 0.1% SDS, 1 % Triton X-100), WB2 (20 mM Tris-HCl pH 8, 2 mM EDTA, 500 mM NaCl, 0.1% SDS, 1 % Triton X-100), WB3 (10 mM Tris-HCl pH 8, 1 mM EDTA, 250 mM LiCl, 1 % deoxycholate, 1 % NP-40), WB4 (10 mM Tris-HCl pH 8, 1mM EDTA) and DNA fragments were eluted in extraction buffer (1% SDS, 0.1 M NaHCO3). For RNA-seq, total RNAs were extracted with the RNeasy Plus kit from QIAGEN including a DNase digestion step, and mRNA were enriched by selection with poly-dT beads. For SCL-exo-seq, genomic DNA was extracted using reagents from the DNeasy Blood and Tissue kit (QIAGEN).
ChIP-seq libraries (2 replicates) were generated with DNA fragments from 6 to 9 pooled individual ChIP experiments and the TruSeq kit from Illumina according to the manufacturer's protocol. For RNA-seq, three replicate libraries of each condition (TruSeq stranded mRNA, Illumina) were prepared and sequenced (single reads of 75 bases) at the Genomic Paris Center facility (Paris, France). For each SCL-exo experiment, 8 micrograms of gDNA were sonicated two times 7 min (30 sec ON /30 sec OFF) and two times 14 min (30 sec ON /30 sec OFF) with a bioruptor (Diagenode). Glucosylation and biotinylation of 5hmC were performed with the Hydroxymethyl Collector kit (Active Motif), followed by on beads-exonuclease digestion of the captured fragments and library preparation (TrueSeq library preparation kit, Ilumina). For normalization purpose, 400 pg of 5hmC Control DNA provided by the Hydroxymethyl Collector kit were added to each sample as spike-in. Libraries from 3 independent SCL-exo experiments were sequenced with a HiSeq 1500 (Illumina) by the GEH facility (Rennes, France).

treatment_protocol: For RNA-seq, ChIP-seq and SCL-exo-seq, cells were kept for 48h in phenol-red-free DMEM (Gibco 31052) supplemented with 2.5% dextran-charcoal-treated fetal calf serum (Eurobio S116365181W), glutamine, sodium pyruvate, non-essential amino acids, Penicillin-Streptomycin and Geneticin at 37°C and 5% CO2 before treatment with 10 nM estradiol or 0.1% EtOH.

value: MCF-7-derived breast cancer cells

tag: genotype/variation

value: TET2 mCD


title: PPD

value: treated with PPD for 48 hours


title: CL53-0h [single-cell RNA-seq]

description:
Single-cell RNA-seq of female ESCs with tetO Xist allele on Bl6 X chromosome.
Mus musculus x Mus castaneous
10X 3'GEX
processed data file: spen_WT_dIDR_dSPOC_10x_ttest.csv

value: ESC

value: WT


title: CL87-24h [single-cell RNA-seq]

description:
Single-cell RNA-seq of delta SPOC-SPEN female ESCs with tetO Xist allele on Bl6 X chromosome, with 24h dox-induced Xist expression.
Mus musculus x Mus castaneous
10X 3'GEX
processed data file: spen_WT_dIDR_dSPOC_10x_ttest.csv

value: KO of SPOC domain of SPEN

value: 24h differentiation in MEF media

value: 24h Dox


title: LysM_stim30.3

value: total RNA


title: E2

value: treated with E2 for 48 hours


title: LysM_unstim.2

value: untreated

value: bone marrow-derived macrophages


title: MCF7_H3K4me3_TET2_CD_Rep2

source_name: TET2 CD cells

value: TET2 CD


title: CLP05-36hAUX-24hDOX-3 [bulk RNA-seq]

description:
Bulk RNA-seq of SPEN-AID degron female ESCs with FL-SPEN rescue construct and tetO Xist allele on Bl6 X chromosome, with 36h AUX treatment and 24h dox-induced Xist expression.
Mus musculus x Mus castaneous
mRNA-seq
processed data file: SPEN_WT_dIDR_rescue_featurecounts.txt
processed data file: spen_WT_KO_dIDR_rescue_bulk_ttest.csv

extract_protocol:
RNA was isolated using the Zymo Research RNA miniprep isolation kit (cat. #50-444-629).
RNA-seq libraries were prepared using the TrueSeq Stranded mRNA Library Prep Kit (Illumina 20020594).

value: female

value: AID-tagged SPEN with full length SPEN rescue

value: tetO-Xist

value: 36h Auxin + 24h Dox


title: Control


title: CLP05-36hAUX-24hDOX-2 [bulk RNA-seq]


title: MCF7_H3K4me3_TET2_mCD_Rep1

value: MCF-7-derived breast cancer cells


title: ko2_1281

city: Montreal

first: Tung

last: Bui

state: Quebec

address: 1160 Pine Ave. W, R507

department: Biochemistry

institute: McGill University

data_processing:
Data filtering - removal of reads containing adaptors, reads containing N > 0.1% (N represents base that could not be determined), and low quality reads (quality score Qscore of over 50% bases of the read is <= 20
Mapping of reads - using STAR method to align reads to mouse genome
Quantification - read count was determined using FPKM method (fragments per kilobase of transcript sequence per millions base pairs sequenced)
Genome_build: Mus musculus mm10
Supplementary_files_format_and_content: Excel file including read counts or FPKM values for each sample, gene ID and gene names

source_name: mammary tumour Beta 1 KO

growth_protocol: Transgenic mice develop mammary tumours, which are collected and flash frozen for subsequent RNA extraction.

extract_protocol:
Frozen pieces of tumours were used for total RNA extraction using RNeasy kit (Qiagen).
Library construction and quality control were done by Novogene, using Illimina HiSeq technology.

treatment_protocol: All famle mice received Doxycycline to enable to inducible transgene system.

value: mammary tumour

value: FVB

value: Beta 1 integrin KO


title: MCF7_H3K4me3_TET2_CD_Rep1


title: MCF7_H3K4me3_EV_Rep2

source_name: EV cells

value: EV


title: CLP05-12hAUX-ESC-1 [bulk RNA-seq]

description:
Bulk RNA-seq of SPEN-AID degron female ESCs with FL-SPEN rescue construct and tetO Xist allele on Bl6 X chromosome, with 12h AUX treatment.
Mus musculus x Mus castaneous
mRNA-seq
processed data file: SPEN_WT_dIDR_rescue_featurecounts.txt
processed data file: spen_WT_KO_dIDR_rescue_bulk_ttest.csv

value: ESC

value: 12h Auxin


title: ko2_4687


title: ko2_9304


title: ko2_29304r


title: ko2_5393


title: MCF7_H3K27me3_TET2_CD_Rep2


title: MCF7_H3K27me3_TET2_mCD_Rep1

value: TET2 mCD


title: CL63-36hAUX-24hDOX-3 [bulk RNA-seq]

description:
Bulk RNA-seq of SPEN-AID degron female ESCs with tetO Xist allele on Bl6 X chromosome, with 36h AUX treatment and 24h dox-induced Xist expression.
Mus musculus x Mus castaneous
mRNA-seq
processed data file: SPEN_WT_dIDR_rescue_featurecounts.txt
processed data file: spen_WT_KO_dIDR_rescue_bulk_ttest.csv

value: AID-tagged SPEN

value: 24h differentiation in MEF media


title: CLP05-12hAUX-ESC-2 [bulk RNA-seq]


title: CLP05-12hAUX-ESC-3 [bulk RNA-seq]


title: MCF7_H3K4me3_EV_Rep1


title: MCF7_H3K27me3_TET2_mCD_Rep2


title: CLP05-36hAUX-24hDOX-1 [bulk RNA-seq]


title: ko2_5399


title: MCF7_H3K27me3_TET2_CD_Rep1


title: CL63-36hAUX-24hDOX-2 [bulk RNA-seq]


title: CL63-36hAUX-24hDOX-1 [bulk RNA-seq]


title: ko2_5091


title: ko2_4686


title: ko2_5100


title: MCF7_H3K27me3_EV_Rep2


title: BRM_KO_CM_scRNA-seq

city: San Francisco

first: Benoit

last: Bruneau

address: 1650 Owens Street

institute: Gladstone Institute of Cardiovascular Disease

data_processing:
Libraries were sequenced on Illumina Novaseq
Raw sequencing reads were converted to FASTQ files using Cellranger 2.0.2 mkfastq
Reads were aligned to mm9  using the Cellranger 2.0.2 count
Cellranger 2.0.2 aggr was used to aggregate libraries from WT and BRM KO cells at normal BMP4 concentration or combined with high BMP4 concentrations
Aggregated gene expression matrix was processed in Seurat 2.3.4. Briefly, cells with UMI counts between 10,000 and 80,000 were retained. Gene expression was normalized, scaled and mitochondrial reads and cell cycle genes were regressed. Cells were clustered based on the top 25-30 prinicipal components.
Genome_build: mm9
Supplementary_files_format_and_content: Processed Seurat expression matrix as a csv file, with columns as cell barcodes and rows as genes

molecule: polyA RNA

source_name: mouse ES cell derived cardiac myocytes

growth_protocol: mouse ESCs were maintained in DMEM media supplemented with LIF and serum

extract_protocol:
Cells on day  10 were singularized using Trypsin 0.25% and 10,000 cells were loaded on a 10X Chromium chip
Chromium Single Cell 3′ GEM, Library & Gel Bead Kit v2 was used to construct libraries

treatment_protocol: mouse ESC lines were differentiated to cardiomyocytes using the Kattman et al 2011 Cardiomyocyte Differentiation protocol.

value: mouse ES cell derived cardiac myocytes

value: Brm-/-

tag: timepoint

value: Day 10


title: MCF7_H3K27me3_EV_Rep1


title: CL63-12hAUX-ESC-2 [bulk RNA-seq]

description:
Bulk RNA-seq of SPEN-AID degron female ESCs with tetO Xist allele on Bl6 X chromosome, with 12h AUX treatment.
Mus musculus x Mus castaneous
mRNA-seq
processed data file: SPEN_WT_dIDR_rescue_featurecounts.txt
processed data file: spen_WT_KO_dIDR_rescue_bulk_ttest.csv

value: female


title: CL63-12hAUX-ESC-3 [bulk RNA-seq]


title: CL63-24hDOX-3 [bulk RNA-seq]

description:
Bulk RNA-seq of SPEN-AID degron female ESCs with tetO Xist allele on Bl6 X chromosome, with 24h dox-induced Xist expression.
Mus musculus x Mus castaneous
mRNA-seq
processed data file: SPEN_WT_dIDR_rescue_featurecounts.txt
processed data file: spen_WT_KO_dIDR_rescue_bulk_ttest.csv

value: 24h Dox


title: CL63-12hAUX-ESC-1 [bulk RNA-seq]

value: tetO-Xist


title: WT_CM_scRNA-seq

value: wild type


title: CL63-24hDOX-1 [bulk RNA-seq]


title: CL63-0hESC-3 [bulk RNA-seq]

description:
Bulk RNA-seq of SPEN-AID degron female ESCs with tetO Xist allele on Bl6 X chromosome.
Mus musculus x Mus castaneous
mRNA-seq
processed data file: SPEN_WT_dIDR_rescue_featurecounts.txt
processed data file: spen_WT_KO_dIDR_rescue_bulk_ttest.csv


title: BRMKO_CM rep 3

data_processing:
Reads were trimmed using the fastq-mcf program (Aronesty, 2011), and then aligned to the mm9 mouse genome assembly using tophat2 (RNA-seq) (Kim et al., 2013). For each gene, reads were counted, and counts-per-million (CPM) calculated using featureCounts (Liao et al., 2014). Finally, edgeR was used to perform differential expression analyses (Robinson et al., 2010).
Genome_build: mm9
Supplementary_files_format_and_content: bigwig

source_name: cardiac myocytes

growth_protocol: ES cells differentiated to cardiac myocytes

extract_protocol:
Total RNA was isolated from 2 mill cells using TRizol Reagent and Qiagen microRNAeasy kit
Libraries were prepared according to NuGEN Ultralow V2 kit and paired-end 100bp sequenced on an Illumina HiSeq 2500 instrument.

value: BRG1-3XFLAG; BRM KO

value: ES cell derived cardiac myocyte

tag: passage


title: BRMKO_CP rep 3

source_name: Cardiac progenitors

value: Es cell derived cardiac progenitor


title: BRMKO_CM rep 2


title: BRMKO_CM rep 1


title: wt_3191

source_name: mammary tumour Beta 1 WT

value: mammary tumour

value: FVB

value: Beta 1 integrin WT


title: BRMKO_CP rep 1


title: BRMKO_CP rep 2


title: wt_784


title: CL63-0hESC-1 [bulk RNA-seq]

value: ESC


title: WT-Syn-4

first: Mark

middle: R

last: Cookson

address: NIHBC 35 - PNRC I BG RM 1A-116

institute: National Institutue on Aging, NIH

description: WT Syn rep 4

data_processing:
Basecalls performed using CASAVA version 1.7.1
STAR_2.4.2a
express-1.5.1
Genome_build: mm10
Supplementary_files_format_and_content: The processed data file includes raw counts of sequencing reads for transcripts that are the output from eXpress, and the processed file is in tab separated text format

source_name: postnatal mouse brain

extract_protocol:
RNA extractions from primary microglia were performed usining to Trizol/Chloroform isolation method. RNA quality was measured using a Agilent 2100 Bioanalyzer RNA 6000 Nano Chip (Agilent) and samples had a mean RNA integrity number of 9.0.
We purified rRNA depleted RNA starting from 1μg total RNA, then synthesized cDNA libraries using TruSeq stranded Total RNA library prep kit, RiboZeroGold (Illumina, RS-122-9008).Before sequencing, cDNA libraries were quantified by digital-PCR using a ddPCR Library quantification kit (Biorad).
cDNA libraries were multiplexed with 4 samples per pool and 7pM of each pool hybridized to a flow cell following cluster generation using an Illumina cluster station. Libraries were sequenced on Illumina HiSeq2500 (Illumina) to generate ~35 million of 100-bp single end reads per library.

value: primary microglia

tag: age

value: post natal day 21

value: alpha-synuclein fibrils


title: BRMKO_MES rep 3

source_name: mesoderm

value: Es cell derived cardiac mesoderm


title: WT-Syn-3

description: WT Syn rep 3


title: CL315-36hAUX-24hDOX-3 [bulk RNA-seq]

description:
Bulk RNA-seq of SPEN-AID degron female ESCs with dIDR-SPEN rescue and tetO Xist allele on Bl6 X chromosome, with 36h AUX and 24h dox-induced Xist expression/differentiation.
Mus musculus x Mus castaneous
mRNA-seq
processed data file: SPEN_WT_dIDR_rescue_featurecounts.txt
processed data file: spen_WT_KO_dIDR_rescue_bulk_ttest.csv

value: AID-tagged SPEN with dIDR-SPEN rescue

value: 24h differentiation in MEF media

value: 36h Auxin + 24h Dox


title: BRMKO_MES rep 2


title: WT-Syn-2

description: WT Syn rep 2


title: WT-Syn-1

description: WT Syn rep 1


title: wt_681


title: wt_287


title: CL63-24hDOX-2 [bulk RNA-seq]

value: female

value: AID-tagged SPEN

value: 24h Dox


title: CL315-36hAUX-24hDOX-2 [bulk RNA-seq]

value: tetO-Xist


title: CL315-36hAUX-24hDOX-1 [bulk RNA-seq]


title: wt_380


title: CL63-0hESC-2 [bulk RNA-seq]


title: BRMKO_MES rep 1


title: wt_2278


title: CL315-12hAUX-ESC-3 [bulk RNA-seq]

description:
Bulk RNA-seq of SPEN-AID degron female ESCs with dIDR-SPEN rescue construct and tetOXist allele on Bl6 X chromosome, with 12h AUX treatment.
Mus musculus x Mus castaneous
mRNA-seq
processed data file: SPEN_WT_dIDR_rescue_featurecounts.txt
processed data file: spen_WT_KO_dIDR_rescue_bulk_ttest.csv

value: 12h Auxin


title: WT_CM rep 3

value: ES cell derived cardiac myocyte


title: WT_CM rep 2


title: WT-LPS-4

description: WT LPS rep 4

value: wild type

value: LPS


title: CL315-12hAUX-ESC-1 [bulk RNA-seq]


title: WT-LPS-6

description: WT LPS rep 6


title: WT_CM rep 1


title: CL315-12hAUX-ESC-2 [bulk RNA-seq]


title: WT-LPS-2

description: WT LPS rep 2


title: WT-LPS-5

description: WT LPS rep 5


title: WT_CP rep 1

value: Es cell derived cardiac progenitor


title: WT-LPS-1

description: WT LPS rep 1


title: LIF-treated BAE rep2

city: San Diego

first: Pin

last: Li

address: 4350 Vision Dr

institute: Moores Cancer Center, UCSD

data_processing:
HTSeq v0.6.1 was used to count the reads numbers mapped to each gene. And then FPKM (expected number of Fragments Per Kilobase of transcript sequence per Millions base pairs sequenced) of each gene was calculated based on the length of the gene and reads count mapped to this gene. Differential expression analysis of two conditions/groups (two biological replicates per condition) was performed using the DESeq R package (1.18.0). The resulting P-values were adjusted using the Benjamini and Hochberg’s approach for controlling the false discovery rate (Benjamini & Hochberg, 2000). Genes with an adjusted p<0.05 found by DESeq were assigned as differentially expressed. FPKM data were then clustered using the log10(FPKM+1) values. Red denotes genes with high expression levels, and blue denotes genes with low expression levels. The color range from red to blue represents log10(FPKM+1) from large to small.
Genome_build: The assembly of the Bos taurus genome Bos_taurus_UMD_3.1 (GenBank assembly accession: GCA_000003055.3) was used as the reference genome. Index of the reference genome was built using Bowtie v2.2.3 and paired-end clean reads were aligned to the reference genome using TopHat v2.0.12.
Supplementary_files_format_and_content: Matrix table with raw gene counts for every gene and every sample

organism: Bos taurus

source_name: bovine aortic endothelial cells

growth_protocol: Bovine choroidal EC (BCE) cells were maintained in low-glucose DMEM supplemented with 10% bovine calf serum (BCS), 2 mM glutamine, 5 ng/ml bFGF and 10ng/ml VEGF on fibronectin-coated culture plates. Bovine aortic endothelial (BAE) cells (P5-P10) were maintained in DMEM-low glucose supplemented with 10% BCS.

extract_protocol:
Total RNAs were isolated using the RNeasy Plus Mini Kit (Qiagene, CAT# 74136) following manufacturer’s instructions and stored at  -80 ℃.
a total amount of 3 μg RNA per sample was used as input material for the RNA sample preparations. Sequencing libraries were generated using NEBNext® Ultra™ RNA Library Prep Kit for Illumina® (NEB, USA) following manufacturer’s recommendations and index codes were added to attribute sequences to each sample.

treatment_protocol: Cells were then incubated in assay medium (for BCE, DMEM-low glucose supplemented with 10% BCS and antibiotics; for BAE, DMEM-low glucose supplemented with 1% BCS and antibiotics) for 3 hours. Cells were subsequently incubated with LIF (10 ng/ml) or vehicle (0.1% BSA in PBS) for 6 hours.

value: BAE cells

value: treated with LIF for 6 hours


title: WT_MES rep 3


title: Basal BCE rep1

source_name: bovine choroidal endothelial cells

value: BCE cells

value: treated with PBS


title: WT_CP rep 2


title: WT-CTR-5

description: WT CTR rep 4

value: primary microglia

value: post natal day 21

value: Control


title: WT-CTR-6

description: WT CTR rep 5


title: WT_CP rep 3


title: Basal BCE rep2


title: WT_MES rep 1

value: Es cell derived cardiac mesoderm


title: LIF-treated BCE rep2


title: WT-CTR-3

description: WT CTR rep 3


title: LIF-treated BAE rep1


title: delta4dus_total_C

data_processing:
For the total RNA samples, the reads were quality- and adaptor-trimmed using trimmomatic 0.36 with options PE ILLUMINACLIP:TruSeq3-PE.fa:2:30:10 LEADING:30 TRAILING:30 SLIDINGWINDOW:4:15 MINLEN:17 AVGQUAL:3.
For the riboseq samples, adaptor sequence were clipped from the reads using  fastx (v0.0.1.4) with options fastx_clipper -l 19 -a "NNNNCTGTAGGCACCATCAAT" -v -n -c -z  -Q 33. The remaining reads were quality trimmed using fastx with options fastx_trimmer -f 2 -l 50 -z -Q 33 and aligned against contaminants (rRNA and tRNA) with bowtie2 (v2.4) with options --sensitive --un-gz.
The filtered reads of both total and riboseq samples were then mapped on S.pombe genome using HISAT2 with default parameters.
Mapped reads were summarized at the gene level using featurecounts.
Genome-wide assessment of differential translations with ribosome profiling data was performed using the R package xtail (v1.1.5) .
Genome_build: ASM294v2.26
Supplementary_files_format_and_content: Comma-separated text files containing the read count per gene for the total and riboseq samples (rna.csv and ribo.csv, respectively), as well as the results of the differential translation analysis performed with xtail (DE_results.csv)

growth_protocol: Cells were grown to mid-log phase without cycloheximide to avoid potential intereference. Cells were then collected on a filter disc in <1 min by filtration.

extract_protocol:
Briefly, ribosome protected fragments were generated by digesting 100 A260 units of cleared lysate with 600 U RNase I (ThermoFisher) at 22°C. The reaction was stopped after 1 h by addition of 15 µl Superase In (ThermoFisher) and loaded on a 10–50% (w/v) sucrose gradient in 50 mM Tris-HCl pH 7.5, 50 mM NH4Cl, 12 mM MgCl2, 0.5 mM DTT, and centrifuged for 4 h at 38 krpm and 4°C in a SW41Ti rotor. Monosomes were fractionated and total RNA isolated. Subsequently, RNA was separated on 15% polyacrylamide gels (8 M urea, 1× TBE buffer) and 28–32 nt ribosome footprints were extracted from the gel.
Purified ribosome protected fragments were cloned with primers that contained four randomized nucleotides to reduce ligation biases and were prepared for strand-specific NGS with the TruSeq Stranded Total RNA Sample Preparation Protocol (Illumina).

treatment_protocol: One third of the cells was immediately set aside in liquid nitrogen (for total RNA preparation used in RNAseq control to establish mRNA abundance). The remaining two-thirds was pulverized under cryogenic conditions in a Planetary Mill PM100 (Retsch) and were further treated to isolate ribosome protected RNAs.

value: C


title: Basal BAE rep2


title: WT-LPS-3

description: WT LPS rep 3


title: WT_MES rep 2


title: WT-CTR-1

description: WT CTR rep 1


title: KO-Syn-5

description: KO Syn rep 5

value: alpha-synuclein fibrils


title: RNA-YwxEyG4_prep2

data_processing:
For smRNA samples: smRNA sequencing reads were trimmed using Trimmomatic (v0.32), removing adaptor, other Illumina-specific sequences and low-quality bases at the end of each read, using a 4bp sliding window to trim where average window quality fell below 30 (phred33 < 30). Trimmed reads with less than 15 bases were discarded. The resulting clean set of reads were then aligned to the drosophila reference genome (dm6) using novoalign (v3.02.12) with a score difference of 0 for calling multiple mapping (-R 0), reporting a maximum of 100 alignments per read (-r All 100), setting miRNA mode (-m 100) and using -l 14 and -t 10,5.5 as alignment scoring parameters.
For RNA samples: RNA sequencing reads were trimmed using Trimmomatic (v0.32), removing adaptor and other Illumina-specific sequences as well as the first four bases from the start of each read, and low-quality bases at the end of each read, using a 4bp sliding window to trim where average window quality fell below 30 (phred33 < 30). Trimmed reads with less than 30 bases were discarded. The resulting clean set of reads were then aligned to the drosophila reference genomes (dm6) using STAR (v2.3.0e) with default parameters. Reads mapping to more than 10 locations in the genome (MAPQ < 1) were discarded.
for ChIP samples: For ChIP-seq data, reads were trimmed of TruSeq adaptor sequences using trimmomatic. Trimmed reads were then aligned toBDGP5 using BWA-MEM, PCR duplicates were soft-clipped using Picard. BigWig files were generated using Homer, and consist of uniquely aligned reads without PCR duplicates.
Genome_build: dm6
Supplementary_files_format_and_content: bigwig files were generated by bedtools (v2.27.1) genomeCoverageBed, using the bam generated by STAR (RNA files) or Novoalign (smRNA files) and a normalization factor equal to the number of mapped reads divided by 100 million.

source_name: 3rd instar larvae eye discs

value: 3rd instar larvae eye discs


title: delta4dus_total_A

value: A


title: WT_D4 [single-cell RNA-seq]

description:
Single-cell RNA-seq of WT ESCs day 4 of EpiLC differentiation.
Mus musculus x Mus castaneous
10X 3'GEX
processed data file: DeltaB_WT_D4_ttest.csv

value: 129S4/SvJae x Cast

value: female

value: ESC (EpiLC)

value: WT

value: 4 day differentiation in EpiLC media


title: delta4dus_total_B

value: B


title: WT_total_C

value: wild-type


title: CLA11-0h [single-cell RNA-seq]

description:
Single-cell RNA-seq of delta IDR-SPEN female ESCs with tetO-Xist allele on Bl6 X chromosome.
Mus musculus x Mus castaneous
10X 3'GEX
processed data file: spen_WT_dIDR_dSPOC_10x_ttest.csv

value: ESC

value: KO of IDR domain of SPEN

value: tetO-Xist


title: WT-CTR-2

description: WT CTR rep 2

value: primary microglia

value: post natal day 21

value: wild type


title: KO-Syn-4

description: KO Syn rep 4


title: KO-Syn-3

description: KO Syn rep 3


title: LIF-treated BCE rep1

value: BCE cells

value: treated with LIF for 6 hours


title: RNA-YwxEyG4_prep3

value: 3rd instar larvae eye discs


title: RNA-YwxEyG4_prep1


title: KO-Syn-2

description: KO Syn rep 2

value: alpha-synuclein fibrils


title: KO-LPS-3

description: KO LPS  rep 3

value: LPS


title: KO-Syn-1

description: KO Syn rep 1


title: WT_total_B


title: WT_total_A

value: A


title: Basal BAE rep1

value: BAE cells

value: treated with PBS


title: RNA-K36MxEyG4_prep3


title: RNA-WTxEyG4_prep3


title: RNA-WTxEyG4_prep2


title: delta4dus_ribo_A

data_processing:
library strategy: Ribo-seq
For the total RNA samples, the reads were quality- and adaptor-trimmed using trimmomatic 0.36 with options PE ILLUMINACLIP:TruSeq3-PE.fa:2:30:10 LEADING:30 TRAILING:30 SLIDINGWINDOW:4:15 MINLEN:17 AVGQUAL:3.
For the riboseq samples, adaptor sequence were clipped from the reads using  fastx (v0.0.1.4) with options fastx_clipper -l 19 -a "NNNNCTGTAGGCACCATCAAT" -v -n -c -z  -Q 33. The remaining reads were quality trimmed using fastx with options fastx_trimmer -f 2 -l 50 -z -Q 33 and aligned against contaminants (rRNA and tRNA) with bowtie2 (v2.4) with options --sensitive --un-gz.
The filtered reads of both total and riboseq samples were then mapped on S.pombe genome using HISAT2 with default parameters.
Mapped reads were summarized at the gene level using featurecounts.
Genome-wide assessment of differential translations with ribosome profiling data was performed using the R package xtail (v1.1.5) .
Genome_build: ASM294v2.26
Supplementary_files_format_and_content: Comma-separated text files containing the read count per gene for the total and riboseq samples (rna.csv and ribo.csv, respectively), as well as the results of the differential translation analysis performed with xtail (DE_results.csv)

tag: molecule subtype

value: ribosome-protected RNA


title: WT_ribo_B

value: B


title: KO-LPS-2

description: KO LPS  rep 2

tag: tissue


title: KO-LPS-4

description: KO LPS  rep 4

tag: genotype


title: KO-CTR-6

description: KO CTR rep 6

tag: treatment

value: Control


title: RNA-K36MxEyG4_prep2


title: delta4dus_ribo_B

tag: replicate


title: delta4dus_ribo_C

value: C


title: WT_ribo_C

value: wild-type


title: WT_ribo_A


title: RNA-WTxEyG4_prep1


title: KO-LPS-1

description: KO LPS  rep 1


title: RNA-K27MxEyG4_prep2


title: RNA-K27MxEyG4_prep3


title: KO-CTR-3

description: KO CTR rep 3


title: E13.5_EC_2Runx1_H3K4me1_input

city: PHILADELPHIA

first: Kai

last: Tan

address: 3501 CIVIC CENTER BLVD

institute: Children's Hospital of Philadelphia

description:
E13_2Runx1_K4me1_IP.bw
E13_2Runx1_K4me1_IP_peaks.bed

data_processing:
ATAC-seq: Sequencing reads were demultiplexed using Bcl2Fastq v2.20 then trimmed and filtered for quality using Trim Galore v0.6.4 with the following settings: fastqc, paired, and trim1. Reads were then aligned to the mouse genome (mm10) using bowtie 2.3.5.1. Only uniquely mapped reads with fewer than 2 mismatches were used for downstream analyses. Samtools v1.1 was used to convert SAM files to BAM files, and Sambamba v0.6.6 was used to filter out duplicates, multi-mappers, reads mapped to ChrM or blacklist regions, and unmapped reads. MACS2 2.1.4 was used for peak calling using the following parameters: BAMPE, q: 0.05. . Merged replicates were used to create bigwig files for visualization using Deeptools v3.3.0 and the following parameters: normalized to reads per genomic content (RPGC), effective genome size: 2308125349, ignore for normalization: ChrX, min fragment length: 20, bin size: 10.
RNA-seq: Sequencing reads were demultiplexed using Bcl2Fastq then trimmed and filtered for quality using Trim Galore v0.6.4 with the following settings: fastqc, paired, and trim1. Reads were then aligned to the mouse genome (mm10) using STAR 2.7.3a and the mm10 gene reference file from UCSC. Only uniquely mapped reads with fewer than 2 mismatches were used for downstream analyses. Samtools v1.1 was used to convert to BAM files, and Sambamba v0.6.6 was used to filter out duplicates, multi-mappers, and unmapped reads. The featureCounts function of the Subread v2.0.0 package was used to extract gene-level and transcript level read counts. EdgeR and TMM normalization were used to calculate normalized RPKM. Bigwig files for visualization were created using Deeptools v3.3.0 from merged BAM files. The following parameters were used: normalized to reads per genomic content (RPGC), effective genome size: 2308125349, ignore for normalization: ChrX, min fragment length: 20, bin size: 10.
ChIP-seq: Sequencing reads were demultiplexed using Bcl2Fastq v2.20 then trimmed and filtered for quality using Trim Galore v0.6.4 with the following settings: fastqc, paired, and trim1. Reads were then aligned to the mouse genome (mm10) using bowtie 2.3.5.1. Only uniquely mapped reads with fewer than 2 mismatches were used for downstream analyses. Samtools v1.1 was used to convert SAM files to BAM files, and Sambamba v0.6.6 was used to filter out duplicates, multi-mappers, reads mapped to ChrM or blacklist regions, and unmapped reads. MACS2 2.1.4 was used for peak calling using the following parameters: broad, q: 0.05. Merged replicates were used to create bigwig files for visualization using Deeptools v3.3.0 and the following parameters: normalized to reads per genomic content (RPGC), effective genome size: 2308125349, ignore for normalization: ChrX, min fragment length: 20, bin size: 10, smooth length: 60, extend reads: 150.
Genome_build: mm10 (GRCm38)
Supplementary_files_format_and_content: BigWig files provided are normalized tracks of merged replicates.
Supplementary_files_format_and_content: Bed files provided are the output of MACS2 peak calling.
Supplementary_files_format_and_content: RPKM_Norm.txt: Tab-delimited text file for RNA-seq samples includes normalized RPKM.

source_name: Endothelial Cells

extract_protocol:
Cells were sorted from mouse fetuses. ChIP reactions were performed using 50,000 FACS-sorted cells per IP. IPed and Input DNA were purified with MinElute PCR Purification Kit.
Libraries were constructed using Rubicom ThruPLEX DNA-Seq Kit. Libraries were size-selected at 100-700 bp by AMPure XP beads. Libraries were quantified with KAPA qPCR and bioanalyzer prior to single-end sequencing.

treatment_protocol: Females were paired with males for timed matings. Females were injected IP with 2mg tamoxifen 24 hours prior to harvesting. Embryos were harvested at the appropriate embryonic day and cells were sorted on the same day as harvest.

value: Endothelial Cells

tag: isolation markers

value: CD41-CD45-Ter119-CD31+VEC+cKIT lo/+

tag: developmental stage

tag: organ

value: whole fetus

value: +2Runx1 (Cre; CKI/CKI)

tag: chip antibody


title: RNA-K36MxEyG4_prep1


title: RNA-K27MxEyG4_prep1


title: KO-CTR-5

description: KO CTR rep 5

tag: strain

value: primary microglia

value: post natal day 21


title: smRNA-YwxEyG4_prep3

value: 3rd instar larvae eye discs


title: E13.5_EC_2Runx1_H3K4me1_rep2

tag: cell type


title: KO-CTR-4

description: KO CTR rep 4