title: Fasted MEP rep1

city: Suzhou

first: Jiawei

last: Qian

state: Jiangsu

address: Renai Road, No. 199 Suzhou University

institute: Cyrus Tang Medical Institute

data_processing:
Sequenced reads were trimmed for adaptor sequence, and masked for low-complexity or low-quality sequence, then mapped to GRCm38 whole genome using hisat2 version 2.2.1.
Mapped reads were then quantified using featureCount version 2.0.1.
Assembly: GRCm38
Supplementary files format and content: raw_gene_count_matrix.csv: Comma-separated values file includes raw read counts for each sample.

source_name: mouse MEP cell

extract_protocol:
Total RNA was extracted from the tissues using TRIzol (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. Approximately 60 mg of tissue was ground into powder in liquid nitrogen in a 2 mL tube, homogenized for 2 minutes and rested horizontally for 5 minutes. The mix was centrifuged for 5 minutes at 12,000×g at 4°C, and then the supernatant was transferred into a new EP tube with 0.3 mL chloroform/isoamyl alcohol (24:1). The mix was shaken vigorously for 15 s and then centrifuged at 12,000×g for 10 minutes at 4°C. After centrifugation, the upper aqueous phase where RNA remained was transferred into a new tube with an equal volume of isopropyl alcohol supernatant and then centrifuged at 13,600 rpm for 20 minutes at 4°C. After deserting the supernatant, the RNA pellet was washed twice with 1 mL 75% ethanol, and then the mix was centrifuged at 13,600 rpm for 3 minutes at 4°C to collect residual ethanol, followed by pellet air drying for 5-10 minutes in a biosafety cabinet. Finally, 25 µL~100 µL of DEPC-treated water was added to dissolve the RNA. Subsequently, total RNA was qualified and quantified using a NanoDrop and Agilent 2100 bioanalyzer (Thermo Fisher Scientific, MA, USA).
Oligo(dT)-attached magnetic beads were used to purify mRNA. Purified mRNA was fragmented into small pieces with fragment buffer at the appropriate temperature. Then, first-strand cDNA was generated using random hexamer-primed reverse transcription, followed by second-strand cDNA synthesis. Afterwards, A-Tailing Mix and RNA Index Adapters were added by incubation for end repair. The cDNA fragments obtained from the previous step were amplified by PCR, and the products were purified by Ampure XP Beads and then dissolved in EB solution. The product was validated on an Agilent Technologies 2100 bioanalyzer for quality control. The double-stranded PCR products from the previous step were heated, denatured and circularized by the splint oligo sequence to obtain the final library. Single strand circle DNA (ssCir DNA) was formatted as the final library. The final library was amplified with phi29 to make DNA nanoballs (DNBs), which had more than 300 copies of one molecule. DNBs were loaded into the patterned nanoarray, and paired-end 100-base reads were generated on the BGISeq500 platform (BGI-Shenzhen, China).

tag: strain

value: megakaryocyte-erythroid progenitor cell

tag: age

value: 8 weeks

tag: genotype

value: 6h fasted


title: Fasted MEP rep3

status: Public on Jan 02 2023

type: SRA

country: China

library_source: transcriptomic

molecule: total RNA

organism: Mus musculus

tag: cell type

value: wild type

tag: treatment


title: Fasted MEP rep2


title: input_rep2_Y14deg_24h

city: Rehovot

first: Anna

last: Uzonyi

address: 234 Herzl street

department: Department of Molecular Genetics

country: Israel

institute: Weizmann Institute of Science

data_processing:
The reads were aligned with STAR/2.7.9a to the hg19 genome. For further analysis, the bam files were filtered for uniquely aligned reads and processed by the bam2Endreads Rscript (Garcia-Campos et al. 2019) for RNA fragment coverage in transcript space.
Assembly: hg19
Supplementary files format and content: Rdata files output of the bam2ReadEnds script of txtools.

molecule: polyA RNA

organism: Homo sapiens

growth_protocol: HEK293T and HEK293T Y14-HA-dTAG cells were maintained in DMEM high glucose (Dutscher) supplemented with 10% of fetal bovine serum (Sigma-Aldrich), and 1% of penicillin and streptomycin (Sigma-Aldrich).

extract_protocol:
RNA was extracted with BIO TRI RNA reagent (Bio-lab)
RNA was poly-A selected with oligo dT-beads (Dynabeads® mRNA DIRECT™ Kit life tech) and subjected to m6Aseq2 according to Dierks et al, 2021 Nature Methods.

treatment_protocol: In 6 well plate, cells were cultivated at 70% confluency and treated with DMSO 1:1000 or 50 nM dTAG13 (Sigma-Aldrich) in DMSO, and harvested after 4, 8, 16 and 24 hours post-treatment.

tag: sample type

value: human

tag: cell line

value: human embryonic kidney

value: 50 nM dTAG13, 24h


title: input_rep2_Y14deg_0h

value: 50 nM dTAG13, 0h


title: Primary human neutrophils, Control, 6h, biol rep 3

city: Montreal

first: Aiten

last: Ismailova

state: Quebec

address: 3655 Promenade Sir William Osler

country: Canada

institute: McGill University

data_processing:
Only RNA samples with OD 260/280 ratio greater than 1.7 and a RNA integrity number (RIN) > 7 were kept for downstream analysis.
These samples were submitted to McGill University and Genome Quebec Innovation Centre for paired-end sequencing at 50M reads by Illumina NovaSeq 6000S2 PE100.
Each RNAseq dataset had similar total number of RNAseq reads sequenced for each condition and total  GC  content. The  Phred  offset  quality  score  was  greater  than  30  for  all  RNAseq  datasets and  the minimum fragment size for alignment was set to 50. These settings yielded 70% to 85% of reads aligning to a gene using the RSubread and EdgeR packages in R. We filtered out genes with low read counts and included genes with counts of 10 or more for at least 1 treatment group (in all replicates) for downstream analysis.
Running the RNAseq quality control pipeline, it was determined that 3 and 2 replicates for control and LPS-treated conditions, respectively, were of high enough  quality  for  downstream  analysis.  We  obtained  read  counts  and  assessed  differential  gene expression using EdgeR and LIMMA R packages, respectively.
Quality control including read trimming and  filtering  out  low  quality  reads,  mapping  to GRCh38 genome  assembly,  summarization  by  gene, normalization and differential gene expression using the R packages EdgeR and DESeq2 was performed at McGill University and Genome Quebec Innovation Centre.
Assembly: GRCh38
Supplementary files format and content: excel file includes output of DESeq2 (differentially expressed genes and their associated log 2 fold changes, log CPM, p- and FDR values).

source_name: Primary human neutrophils

extract_protocol:
Total RNA was extracted using FavorPrep Blood/Cultured Cell Total RNA Mini Kit (FABRK 001, Favorgen). 500 ng of total RNA was used for the construction of sequencing libraries.
RNA libraries for RNAseq were prepared using NEBNext dual mRNASeq stranded library kit following manufacturer's protocol.

value: Primary human neutrophils

tag: time point

value: vehicle


title: input_rep1_WTdeg_4h

value: DMSO, 4h


title: WT MEP rep2

value: control


title: WT MEP rep1


title: Mouse TA, matrix gel treated 3

city: Fayetteville

first: Jeffrey

last: Wolchok

state: AR

address: 700 W. Research Blvd

department: Biomedical Engineering

country: USA

institute: University of Arkansas

data_processing:
usegalaxy.eu pipeline
Use RNA-STAR for Gapped-read mapper for RNA-seq data ( input 2 pair-end raw files per sample)
Use feature to measure gene expression in RNA-Seq experiments from BAM files ( generated from RNA-Star)
Use edgeR to perform differential expression of count data
Assembly: mm39
Supplementary files format and content: tabular  files include normalized counted reads, per gene for each sample

source_name: Tibialis anterior muscle

extract_protocol:
RNA extarction was are performed by comerical lab BGI
RNA libraries were constructed by comerical lab BGI

treatment_protocol: 50 µl of matrix gel (6 mg/ml) on the left TA, and 50 µl of vehicle control (PBS) on the right TA

tag: tissue

value: Tibialis anterior muscle

value: matrix gel


title: fem1-1_endosperm_sRNA

city: Nanjing

first: Lili

last: Wang

address: Weigang No.1

institute: Nanjing Agricultural University

data_processing:
Adapter sequences were trimmed and clean reads with sizes ranging from 18 to 45 were mapped to MSU7.0 using Bowtie with parameter -v 0.
Annotated structure RNAs were excluded.
Read counts were normalized to RPTM.
Genome_build: MSU7.0
Supplementary_files_format_and_content: tdf

organism: Oryza sativa Japonica Group

growth_protocol: Rice plants grown in soil in jiangsu province

extract_protocol:
RNA was extrated using TRIzol method.
18-45nt small RNA was cut from PAGE gel.

tag: cultivar

value: endosperm


title: input_rep1_WTdeg_16h

value: DMSO, 16h


title: WT MEP rep3


title: TP309_endosperm_sRNA


title: input_rep2_WTdeg_0h

value: DMSO, 0h


title: input_rep1_Y14deg_8h

value: 50 nM dTAG13, 8h


title: pol iv_seedcoat_sRNA

source_name: pol iv_seedcoat

value: seedcoat

value: pol iv


title: pol iv_endosperm_sRNA

source_name: pol iv_endosperm


title: input_rep1_WTdeg_0h


title: input_rep1_Y14deg_16h

value: 50 nM dTAG13,16h


title: input_rep1_Y14deg_4h

value: human

value: 50 nM dTAG13,4h


title: fem1-1_seedcoat_sRNA


title: input_rep1_Y14deg_0h

value: human embryonic kidney


title: Nol12_noint_rep2_IP_integrated

data_processing:
Reads were mapped to hg19 genome assembly and the expressed stable integration sequences with STAR/2.7.9a (Dobin et al., 2013). Per-base read coverages were obtained with txtools (https://github.com/AngelCampos/txtools)
Assembly: hg19 and integrated sequences
Supplementary files format and content: R data table (rds) file output from txtools https://github.com/AngelCampos/txtools. In addition, the TableS1.xlsx excel file contains the expressed integrated sequences.

growth_protocol: MCF7 cells (ATCC) were maintained in DMEM (GIBCO) supplemented with 10% FBS and 1% Penicillin and Streptomycin.

treatment_protocol: For stable expression of reporter genes, MCF7 cells bearing a genome-integrated Flp-In site were transfected with pcDNA5/FRT/TO-encoded genes and pOG44 recombinase in a 1:9 ratio. After 48 hours, the medium was supplemented with Hygromycin at 0.1mg/ml final concentration and grown for three weeks with weekly change of medium.

value: human breast cancer

value: Stable integration of MR3.


title: Nol12_noint_rep1_IP_integrated


title: Nip_branch_sRNA

description: Nip_branch sRNA-seq

data_processing:
Adapter sequences were trimed and clean reads with sizes ranging from 18 to 45 were mapped to MSU7.0 using Bowtie with parameter -v 0.
Annotated structure RNAs were excluded.
Read counts were normalized to RPTM.
Genome_build: MSU7.0

source_name: small RNA

extract_protocol:
RNA was extrated using TRIzol method.
18-30nt small RNA was cut from PAGE gel.

value: branch

value: Nipponbare

value: wildtype


title: TP309_panicle_R2_sRNA

description: TP309_panicle_R2 sRNA-seq

value: panicle


title: input_rep1_WTdeg_8h

value: DMSO, 8h


title: input_rep1_WTdeg_24h


title: TP309_panicle_R1_sRNA

description: TP309_panicle_R1 sRNA-seq


title: Nol12_noint_rep1_input_integrated


title: Nol12_mut_rep2_input_integrated

value: Stable integration of MR2.


title: Nol12_mut_rep2_IP_integrated


title: TP309_seedcoat_sRNA


title: input_rep1_Y14deg_24h

value: 50 nM dTAG13, 24h


title: control_actD_9h_rep2

description:
HEK293T cells treated with Mettl3 inhibitor or untreated, and treated with actD for 0,3,6,9h.
actD_Mettl3inh.bed

data_processing:
Reads were mapped to hg19 genome annotation using STAR/2.7.9a (Dobin et al., 2013). Total read count per gene was computed with bedtools multicov (Quinlan and Hall 2010), with the samples ordered as non-treated 0 hours replicates, 3 hours replicates, 6 hours replicates, 9 hours replicates, followed by the treated samples ordered similarly.
Assembly: hg19
Supplementary files format and content: Bed file with read quantification, ordered as non-treated 0 hours replicates, 3 hours replicates, 6 hours replicates, 9 hours replicates, followed by the treated samples ordered similarly.

growth_protocol: HEK293T cells were cultured in DMEM (Gibco) supplemented with 10% FBS and 1% Penicillin and Streptomycin.

extract_protocol:
RNA was extracted with BIO TRI RNA reagent (Bio-lab)
RNA was poly-A selected with oligo dT-beads (Dynabeads® mRNA DIRECT™ Kit life tech) , and libraries were prepared from the full transcriptome.

treatment_protocol: For treatment with Actinomycin D the cells were seeded into 60-mm dishes and grown for one day until 50% confluent.  Actinomycin D was added (7.5µM) for the indicated time points.

value: non-treated samples related to the Mettl3 inhibitor STM2457 treatment (15h), actD treatment 9h, rep2


title: M25 Rat skeletal muscle sample at 24 h of postmortem interval

type: RNA

city: Ciudad de México

first: Mariano

last: Guardado Estrada

state: Ciudad de México

address: Circuito de la Investigación SN

country: Mexico

institute: UNAM

hyb_protocol: Labeled samples were hybridized with Affymetrix GeneChip miRNA 4.0 Array (Affymetrix, Santa Clara, CA, USA).

scan_protocol: Affymetrix Gene ChIP Scanner 3000 7G

data_processing: The CEL files were analyzed with the Affymetrix Transcriptome Analysis Console (TAC) Software. With this software, the probes intensities of the miRNAs were nor-malized, and the miRNA gene expression was calculated with the Robust Multi-chip Analysis (RMA), setting the value of Detected Above Background (DABG) to 0.05.

label: biotin

organism: Rattus norvegicus

source_name: Rat skeletal muscle sample

label_protocol: Samples were biotin labeled with the Affymetrix® FlashTag™ Biotin HSR RNA Labeling Kit according to the manufacturer protocol.

extract_protocol: 200 mg were processed with Trizol to extract total RNA

value: femur muscle

value: Shh -/-


title: M24 Rat skeletal muscle sample at 24 h of postmortem interval


title: M26 Rat skeletal muscle sample at 24 h of postmortem interval


title: Nip_bract_sRNA

description: Nip_bract sRNA-seq

value: bract (palea and lemma)


title: Nol12_noint_rep2_input_integrated


title: M23 Rat skeletal muscle sample at 24 h of postmortem interval

value: wild-type


title: Nol12_mut_rep1_input_integrated

value: human


title: control_actD_9h_rep1

value: non-treated samples related to the Mettl3 inhibitor STM2457 treatment (15h), actD treatment 9h, rep1


title: control_actD_6h_rep1

value: human embryonic kidney

value: non-treated samples related to the Mettl3 inhibitor STM2457 treatment (15h), actD treatment 6h, rep1


title: control_actD_3h_rep2

value: non-treated samples related to the Mettl3 inhibitor STM2457 treatment (15h), actD treatment 3h, rep2


title: M3 Rat skeletal muscle sample


title: M22 Rat skeletal muscle sample at 24 h of postmortem interval


title: M6 Rat skeletal muscle sample


title: M4 Rat skeletal muscle sample


title: control_actD_0h_rep2

value: non-treated samples related to the Mettl3 inhibitor STM2457 treatment (15h), actD treatment 0h, rep2


title: Nol12_mut_rep1_IP_integrated

value: human breast cancer


title: M2 Rat skeletal muscle sample


title: RLT11

city: Seoul

first: Jungmin

last: Choi

state: Seoul

address: 73, Goryeodae-ro, Seongbuk-gu

department: Department of Biomedical Sciences

country: South Korea

institute: Korea University College of Medicine

data_processing:
Sequencing reads were trimmed for bases with base quality score less than 20
Trimmed sequencing reads were aligned to human transcriptome mm10 using HISAT2
Mapped reads were converted to the count table using StringTie2
Assembly: mm10
Supplementary files format and content: comma-delimited text file containing the raw count data for all samples

source_name: Bone marrow mesenchymal stem cells from mice transplanted with ALL and treated with LTbR-Ig.

extract_protocol:
Long bones were flushed with HBSS supplemented with 2% of heat-inactivated fetal bovine serum, Penicillin/Streptomycin, L-glutamine, HEPES, and 200 U/mL Collagenase IV (Worthington Biochemical Corporation) and digested for 30 min at 37°C. Cell clumps were dissociated by gentle pipetting. Cells were filtered through a 100 mm nylon mesh and washed with HBSS/2% FBS. Hematopoietic cells were depleted by staining with biotin-conjugated CD45 and Ter119 antibodies, and using Dynabeads ® Biotin Binder (Invitrogen #11047). Following depletion of hematopoietic cells, the remaining cells were stained with antibodies against CD31, CD144 and PDGFRb. BM MSCs were identified as CD45- Ter119- CD31- CD144- PDGFRb+ cells. Sorting was performed using a BD FACS Aria II. Cells were sorted directly into 350 mL RLT plus buffer (Qiagen) and RNA extracted using the RNeasy® Plus Micro Kit (Qiagen #74034). RNA sequencing was performed by the Yale Center for Genome Analysis using the Illumina HiSeq2500 system, with paired-end 2 x 76 bp read length.
Libraries were prepared after poly-A selection

value: Bone marrow

value: Mesenchymal stem cells

value: ALL treated with LTbR-Ig

value: ALL(LTbR-lg)


title: control_actD_3h_rep1

value: non-treated samples related to the Mettl3 inhibitor STM2457 treatment (15h), actD treatment 3h, rep1


title: RLT8

source_name: Bone marrow mesenchymal stem cells from mice transplanted with ALL and treated with Hel-Ig.

value: ALL treated with Hel-Ig

value: ALL(Hel-lg)


title: RLT10


title: control_actD_0h_rep1

value: non-treated samples related to the Mettl3 inhibitor STM2457 treatment (15h), actD treatment 0h, rep1


title: RLT6


title: AS_F_2

city: Cleveland

first: Ernest

middle: R

last: Chan

state: OH

address: 10900 Euclid Ave, Robbins E211

department: PQHS

institute: CWRU

data_processing:
Illumina reads were mapped to reference genome using STAR Aligner program.
The expression levels of genes were determined using Cufflinks and presented as FPKM
Differential expression analysis was done using Cuffdiff
Assembly: hg19
Supplementary files format and content: matrix.txt (tab delimited file containing FPKM values for each gene/sample)

source_name: Peripheral blood

extract_protocol:
total RNA was extracted as per Takara kit
RNA libraries were prepared for sequencing using standard Illumina protocols

treatment_protocol: Stimulation of PBMCs with CytoSim for 4 hours then enrichment by interleukin (IL)-17-phycoerythrin (PE) cytokine secretion assay

tag: disease state

value: Ankylosing Spondylitis (AS)

tag: Sex

value: F

value: Peripheral blood

value: PBMCs

value: Cytostim for 4 hours


title: control_actD_6h_rep2

value: human

value: human embryonic kidney

value: non-treated samples related to the Mettl3 inhibitor STM2457 treatment (15h), actD treatment 6h, rep2


title: RLT7


title: Calm3_distToPolyA_68_IP_rep2

description:
MCF7 cells transfected with Calm3 contruct with 68nt distance between the DRACH motif and polyA site, IP, rep2
TableS1.xlsx

data_processing:
Reads were mapped to hg19 genome assembly and the transfected plasmid sequences with STAR/2.7.9a (Dobin et al., 2013). Per-base read coverages were obtained with txtools (https://github.com/AngelCampos/txtools)
Assembly: hg19 and the transfected plasmid (See TableS1 for sequence)
Supplementary files format and content: R data table (rds) file output from txtools https://github.com/AngelCampos/txtools. In addition, the TableS1.xlsx excel file contains the tranfected plasmid sequences.

treatment_protocol: Transfections were done with JetPrime (Polyplus) reagent, according to the supplier’s instructions. Typically, the cells were seeded in 6-cm dishes (Corning) and on the next day transfected with 1-2µg of plasmids. The cells were harvested after additional 24 hours by scraping and subjected to RNA extraction.

value: human cell line, plasmid with mouse sequence

value: human breast cancer

value: Plasmid transfection with an insert described in the 'description'. Insert sequences are in TableS1.xlsx.


title: RLT4

source_name: Bone marrow mesenchymal stem cells from untreated control mice.

value: Bone marrow

value: Mesenchymal stem cells

value: WT

value: Control


title: RLT12

value: ALL treated with LTbR-Ig

value: ALL(LTbR-lg)


title: AS_F_6

value: Ankylosing Spondylitis (AS)


title: RLT2


title: RLT5

value: ALL treated with Hel-Ig

value: ALL(Hel-lg)


title: Calm3_distToPolyA_50_input_rep1

description:
MCF7 cells transfected with Calm3 contruct with 50nt distance between the DRACH motif and polyA site, input, rep1
TableS1.xlsx


title: MCF10A, TGFb3, rep1


title: Calm3_distToPolyA_25_IP_rep2

description:
MCF7 cells transfected with Calm3 contruct with 25nt distance between the DRACH motif and polyA site, IP, rep2
TableS1.xlsx


title: Calm3_distToPolyA_50_IP_rep2

description:
MCF7 cells transfected with Calm3 contruct with 50nt distance between the DRACH motif and polyA site, IP, rep2
TableS1.xlsx


title: ChIP-BS-seq_TSC1_WT_H3K27me3_Rep2

city: Berlin

first: Sara

last: Hetzel

address: Ihnestraße 63

department: Genome Regulation

country: Germany

institute: Max Planck Institute for Molecular Genetics

library_source: genomic

data_processing:
Raw reads of ESC and TSC H3K27me3 ChIP-BS-seq samples as well as their respective input samples were subjected to adapter and quality trimming with cutadapt (version 2.4; parameters: --quality-cutoff 20 --overlap 5 --minimum-length 25 --adapter AGATCGGAAGAGC -A AGATCGGAAGAGC).
Reads were aligned to the mouse genome (mm10) using BSMAP (version 2.90; parameters: -v 0.1 -s 16 -q 20 -w 100 -S 1 -u -R).
A sorted BAM file was obtained and indexed using samtools with the ‘sort’ and ‘index’ commands (version 1.10).
Duplicate reads were identified and removed using GATK (version 4.1.4.1) ‘MarkDuplicates’ and default parameters.
Replicates of treatment and input samples were merged respectively using samtools ‘merge’.
Methylation rates were called using mcall from the MOABS package (version 1.3.2; default parameters).
All analyses were restricted to autosomes and only CpGs covered by at least 10 and at most 150 reads were considered for downstream analyses.
Genome-wide coverage tracks for single and merged replicates normalized by library size were computed using deepTools bamCoverage (parameters: --normalizeUsing RPGC --extendReads).
Coverage tracks were subtracted by the respective input using deeptools ‘bigwigCompare’.
Assembly: mm10
Supplementary files format and content: H3K27me3: Bigwig coverage files (RPGC normalization, input subtracted).
Supplementary files format and content: DNA methylation: Bigwig file containing methylation rates for CpGs covered by at least 10 and at most 150 reads: <chr> <start> <end> <methylation rate>
Library strategy: ChIP-BS-seq

molecule: genomic DNA

source_name: Trophoblast stem cells

growth_protocol: TSCs (TSC line 1) were cultured in TSC medium (RPMI+GlutaMAX (Thermo, #61870044), 20 % fetal bovine serum (PAN, #P30-2602), 1 mM sodium pyruvate (Thermo, #11360070), 100 µM 2-mercaptoethanol (Thermo, #21985023) and 1X Penicillin/Streptomycin (Thermo, #15140122); 25 ng/ml FGF4 (R&D systems, #235-F4-025) and 1 µg/ml Heparin (Sigma, #H3149).

extract_protocol:
5 million cells were resuspended in 500 µl cell lysis buffer (20 mM Tris-HCl ph8, 85 mM KCl, 0.5% NP40) and incubated for 5 minutes on ice followed by 2500 g centrifugation at 4 ºC for 5 minutes. Supernatant was removed and pelleted nuclei were resuspended in 100 µl PBS, after which an additional 100 µl of 2x lysis buffer supplemented with 40 U/µl micrococcal nuclease (NEB) was added (100 mM Tris-HCL pH 8.0, 300 mM NaCl, 2% TritonX-100, 0.2% sodium deoxycholate, and 10 mM CaCl2). Nuclear lysis was carried out on ice for 20 minutes followed by a 25 minute incubation at 37 ºC, which was previously experimentally determined to yield mono-nucleosome sized fragments. The micrococcal nuclease reaction was terminated with the addition of 800 µl lysis dilution buffer (50 mM Tris-HCl pH 8.0, 150 mM NaCl, 1% TritonX-100, 50 mM EGTA, 50 mM EDTA, and 0.1% sodium deoxycholate) and 2 µg of H3K27me3 antibody was added (Thermo Scientific MA5-11198). Immunoprecipitation was carried out at 4 ºC overnight with gentle rotation followed by incubation with protein A dynabeads (Thermo Scientific) for 4 hours the next day. Bead bound immune complexes were washed at 4 ºC two times with RIPA buffer (0.1% DOC, 0.1% SDS, 1% Triton X-100, 10mM Tris-HCl pH 8.0, 1mM EDTA, 140 Mm NaCl), and then one time with each of the following: RIPA high salt (0.1% DOC, 0.1% SDS, 1% Triton X-100, 10mM Tris-HCl pH 8.0, 1mM EDTA, 360 mM NaCl), LiCl wash buffer (250mM LiCl, 0.5% NP40, 0.5% deoxycholate, 1mM EDTA, 10mM Tris-HCl, pH 8.0), and TE pH 8.0. DNA was then eluted from the protein A beads by dissolving them in 100 µL ChIP elution buffer (TE, 0.1% SDS, and 300mM NaCl) with 0.2 mg/mL Proteinase K (Invitrogen) and incubating at 55 ºC overnight. The next day 300 µl TE was added to the reaction along with 400 µl phenol-chloroform-isoamyl alcohol (Invitrogen), the solution was briefly vortexed, and then centrifuged at 21,000 g for 10 minutes at room temperature in phase lock tubes (VWR). After centrifugation, the aqueous phase was collected and combined with 20 µL 5M NaCl, 1 µL 20 mg/mL glycogen (Thermo Scientific), and 880 µL 100% ethanol. This solution was stored at -20 ºC overnight and then centrifuged at 21,000 g 4 ºC for 1 hour the next day. This was followed by two washes with 70% ethanol and eluted in 20 µL 1x TE.
The resulting DNA was bisulfite converted with the EZ DNA methylation gold kit (Zymo) and used as input for the Accel-NGS® Methyl-Seq DNA Library Kit (Swift/IDT) following the manufacturer’s protocol. All libraries were sequenced using 100 bp paired-end sequencing (200 cycles kit) on a NovaSeq 6000 platform.

value: Trophoblast stem cells

tag: antibody


title: Calm3_distToPolyA_50_input_rep2

description:
MCF7 cells transfected with Calm3 contruct with 50nt distance between the DRACH motif and polyA site, input, rep2
TableS1.xlsx


title: ChIP-BS-seq_TSC1_WT_H3K27me3_Rep1


title: Calm3_distToPolyA_50_IP_rep1

description:
MCF7 cells transfected with Calm3 contruct with 50nt distance between the DRACH motif and polyA site, IP, rep1
TableS1.xlsx


title: Calm3_distToPolyA_68_input_rep1

description:
MCF7 cells transfected with Calm3 contruct with 68nt distance between the DRACH motif and polyA site, input, rep1
TableS1.xlsx


title: Calm3_distToPolyA_25_input_rep2

description:
MCF7 cells transfected with Calm3 contruct with 25nt distance between the DRACH motif and polyA site, input, rep2
TableS1.xlsx


title: ChIP-BS-seq_TSC1_WT_H3K27me3_Input_Rep2


title: ChIP-BS-seq_TSC1_WT_H3K27me3_Input_Rep1


title: ChIP-BS-seq_ESC_WT_H3K27me3_Rep2

source_name: Embryonic stem cells

growth_protocol: ESCs were cultured in Knockout DMEM (Thermo, #10829018, 15% fetal bovine serum (PAN, #P30-2602), 1X GlutaMAX supplement (Thermo, #35050-038), 1X non-essential amino acids (Thermo, #11140-035), 100 µM 2-mercaptoethanol (Thermo, #21985023), 1X Penicillin/Streptomycin (Thermo, #15140122) and leukemia inhibitory factor (LIF)

value: Embryonic stem cells


title: Calm3_distToPolyA_25_IP_rep1

description:
MCF7 cells transfected with Calm3 contruct with 25nt distance between the DRACH motif and polyA site, IP, rep1
TableS1.xlsx


title: Calm3_distToPolyA_25_input_rep1

description:
MCF7 cells transfected with Calm3 contruct with 25nt distance between the DRACH motif and polyA site, input, rep1
TableS1.xlsx


title: ChIP-BS-seq_ESC_WT_H3K27me3_Rep1


title: ChIP-BS-seq_ESC_WT_H3K27me3_Input_Rep1


title: ChIP-BS-seq_ESC_WT_H3K27me3_Input_Rep2


title: Calm3_distToPolyA_68_IP_rep1

description:
MCF7 cells transfected with Calm3 contruct with 68nt distance between the DRACH motif and polyA site, IP, rep1
TableS1.xlsx


title: Calm3_distToPolyA_68_input_rep2

description:
MCF7 cells transfected with Calm3 contruct with 68nt distance between the DRACH motif and polyA site, input, rep2
TableS1.xlsx


title: Calm3_distToPolyA_200_input_rep2

description:
MCF7 cells transfected with Calm3 contruct with 200nt distance between the DRACH motif and polyA site, input, rep2
TableS1.xlsx


title: Calm3_distToPolyA_200_IP_rep2

description:
MCF7 cells transfected with Calm3 contruct with 200nt distance between the DRACH motif and polyA site, IP, rep2
TableS1.xlsx


title: ChIPseq_TSC1_WT_EZH2i_5w_4d_EED_Rep1

data_processing:
Raw reads of ESC and TSC EED ChIP-seq samples as well as their respective input samples were subjected to adapter and quality trimming with cutadapt (version 2.4; parameters: --quality-cutoff 20 --overlap 5 --minimum-length 25 --adapter AGATCGGAAGAGC -A AGATCGGAAGAGC).
Reads were aligned to the mouse genome (mm10) using BWA with the ‘mem’ command (version 0.7.17, default parameters).
A sorted BAM file was obtained and indexed using samtools with the ‘sort’ and ‘index’ commands (version 1.10).
Duplicate reads were identified and removed using GATK (version 4.1.4.1) ‘MarkDuplicates’ and default parameters.
Replicates of treatment and input samples were merged respectively using samtools ‘merge’.
Peaks were called using MACS2 ‘callpeak’ (version 2.1.2; parameters --bdg --SPMR --broad) based on merged replicates using the input samples as control samples and only peaks with a q-value < 0.01 were considered for downstream analyses.
Genome-wide coverage tracks for single and merged replicates normalized by library size were computed using deepTools bamCoverage (parameters: --normalizeUsing RPGC --extendReads).
Coverage tracks were subtracted by the respective input using deeptools ‘bigwigCompare’.
Assembly: mm10
Supplementary files format and content: Bigwig coverage files (RPGC normalization, input subtracted).

extract_protocol:
10 million cells were crosslinked in a 1% formaldehyde solution for 5 minutes at room temperature, after which glycine was added to a final concentration of 125 mM and incubated for 5 minutes to quench the reaction. These fixed cells were centrifuged at 2,500 g for 5 minutes at 4 °C and the pellet was washed twice with 1 mL PBS. Nuclei were extracted by incubating the fixed cells with 500 µL of cell lysis buffer (20 mM Tris-HCl pH 8.0, 85 mM KCl, 0.5% NP40) for 10 min on ice then spun down for 3 min at 2,500 g. The pellet was resuspended in nuclei lysis buffer (10 mM Tris pH7.5, 1% IGEPAL, 0.5% sodium deoxycholate, 0.1% SDS) then sonicated on a Covaris E220 Evolution sonicator (peak incident power 140.0, duty factor 5.0, cycles per burst 200, 20 min). After sonication, chromatin was spun down at 21,000 g for 10 min to pellet insoluble material. The supernatant was transferred to a fresh tube, the volume was increased to 1 ml with chip dilution buffer (0.01% SDS, 1.1% Triton X-100, 1.2 mM EDTA, 16.7 mM Tris-HCl pH 8.1, 167 mM NaCl), and 5 µg of EED antibody was added (Abcam ab240650). Immunoprecipitation was carried out at 4 ºC overnight with gentle rotation followed by incubation with protein A dynabeads (Thermo Scientific) for 4 hours the next day. This was followed by two washes of each of the following: low salt wash buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl pH 8.1, 150 mM NaCl); high salt wash buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris, pH 8.1, 500 mM NaCl); LiCl wash buffer (0.25 M LiCl, 1% NP40, 1% deoxycholate, 1 mM EDTA, 10 mM Tris-HCl pH 8.1) and TE buffer pH 8.0 (10 mM Tris-HCl, pH 8.0, 1 mM EDTA pH 8.0). DNA was eluted twice using 50 µl of elution buffer (0.5–1% SDS and 0.1 M NaHCO3) at 65 °C for 15 min. A 16-µl volume of reverse crosslinking salt mixture (250 mM Tris-HCl, pH 6.5, 62.5 mM EDTA pH 8.0, 1.25 M NaCl, 5 mg ml−1 Proteinase K) was added, and samples were allowed to incubate at 65 °C overnight.  The next day 284 µl TE was added to the reaction along with 400 µl phenol-chloroform-isoamyl alcohol (Invitrogen), the solution was briefly vortexed, and then centrifuged at 21,000 g for 10 minutes at room temperature in phase lock tubes (VWR). After centrifugation, the aqueous phase was collected and combined with 20 µL 5M NaCl, 1 µL 20 mg/mL glycogen (Thermo Scientific), and 880 µL 100% ethanol. This solution was stored at -20 ºC overnight and then centrifuged at 21,000 g 4 ºC for 1 hour the next day. This was followed by two washes with 70% ethanol and elution of the pelleted DNA in 50 µL 1x TE.
Libraries were prepared using NEBNext Ultra II DNA Library Prep Kit for Illumina (NEB) following the manufacturer’s protocol. All libraries were sequenced using 100 bp paired-end sequencing (200 cycles kit) on a NovaSeq 6000 platform.

treatment_protocol: TSCs (TSC line 1) were cultured in TSC medium (RPMI+GlutaMAX (Thermo, #61870044), 20 % fetal bovine serum (PAN, #P30-2602), 1 mM sodium pyruvate (Thermo, #11360070), 100 µM 2-mercaptoethanol (Thermo, #21985023) and 1X Penicillin/Streptomycin (Thermo, #15140122); 25 ng/ml FGF4 (R&D systems, #235-F4-025) and 1 µg/ml Heparin (Sigma, #H3149) for 5 weeks and 4 days with the addition of EZH2 inhibitor; dissolved in DMSO to 1 mM) at a final concentration of 1 µM.

value: EED


title: Calm3_distToPolyA_200_input_rep1

description:
MCF7 cells transfected with Calm3 contruct with 200nt distance between the DRACH motif and polyA site, input, rep1
TableS1.xlsx


title: Calm3_distToPolyA_200_IP_rep1

description:
MCF7 cells transfected with Calm3 contruct with 200nt distance between the DRACH motif and polyA site, IP, rep1
TableS1.xlsx


title: Calm3_distToPolyA_150_IP_rep2

description:
MCF7 cells transfected with Calm3 contruct with 150nt distance between the DRACH motif and polyA site, IP, rep2
TableS1.xlsx


title: ChIPseq_TSC1_WT_EZH2i_5w_4d_EED_Input


title: ChIPseq_TSC1_WT_EED_Rep2

treatment_protocol: No treatment


title: ChIPseq_TSC1_WT_EZH2i_5w_4d_EED_Rep2


title: ChIPseq_TSC1_WT_EED_Rep1


title: Calm3_distToPolyA_150_IP_rep1

description:
MCF7 cells transfected with Calm3 contruct with 150nt distance between the DRACH motif and polyA site, IP, rep1
TableS1.xlsx


title: Calm3_distToPolyA_100_input_rep2

description:
MCF7 cells transfected with Calm3 contruct with 100nt distance between the DRACH motif and polyA site, input, rep2
TableS1.xlsx


title: ChIPseq_TSC1_WT_EED_Input_Rep2


title: Calm3_distToPolyA_100_IP_rep2

description:
MCF7 cells transfected with Calm3 contruct with 100nt distance between the DRACH motif and polyA site, IP, rep2
TableS1.xlsx


title: Calm3_distToPolyA_100_IP_rep1

description:
MCF7 cells transfected with Calm3 contruct with 100nt distance between the DRACH motif and polyA site, IP, rep1
TableS1.xlsx


title: ChIPseq_ESC_WT_EED_Rep1


title: cDNA_pLenti6_pool3_IP_rep2

description:
NIH3T3 cells transfected with a pool of 12 cDNA pLenti6 plasmids.
cDNAExpV2IPpool3_AlignedReads.bam.txDT.rds
TableS1.xlsx

data_processing:
Reads were mapped to mm9 genome and corresponding cDNA sequences (Table S1) using STAR/2.7.9a (Dobin et al., 2013).
Assembly: mm9
Supplementary files format and content: R data table (rds) file output from txtools https://github.com/AngelCampos/txtools. In addition, the TableS1.xlsx excel file contains the tranfected plasmid sequences. Replicates of the same pool were merged together.

growth_protocol: NIH3T3 cells were cultured in DMEM (Gibco) supplemented with 10% FBS and 1% Penicillin and Streptomycin.

extract_protocol:
RNA was extracted with NucleoZOL (Macherey-Nagel)
RNA was poly-A selected with oligo dT-beads (Dynabeads® mRNA DIRECT™ Kit life tech) and subjected to m6Aseq2 according to Dierks et al, 2021 Nature Methods.

treatment_protocol: Cells were seeded on 60 mm plates 24 hours prior to transfection. Cells were transfected with pLenti cDNA constructs with Lipofectamine 2000 (Thermo Fisher Scientific) reagent according to the manufacturer’s protocol.  A total of 33 different cDNA plasmids were transfected in pools of 12, in duplicates.

value: mouse, human

value: mouse fibroblast

value: Plasmid transfection with cDNA pLentli6 plasmid pool described in Table S1.


title: ChIPseq_ESC_WT_EED_Input_Rep1


title: cDNA_pLenti6_pool2_IP_rep2

description:
NIH3T3 cells transfected with a pool of 12 cDNA pLenti6 plasmids.
cDNAExpV2IPpool2_AlignedReads.bam.txDT.rds
TableS1.xlsx


title: AKO_6

city: Madison

first: Sean

middle: M

last: Ronnekleiv-Kelly

state: Wisconsin

address: 1400 University Ave

department: Surgery

institute: University of Wisconsin

data_processing:
Illumina bcl2fastq v2.20.0.422
Read trimming with Skewer v0.2.2b -m pe -q 20 -k 15 -l 25
Read alignment with STAR v2.5.3a --runThreadN 10 --twopassMode Basic --quantMode TranscriptomeSAM GeneCounts --outSAMtype BAM SortedByCoordinate --outSAMstrandField intronMotif --outSAMattributes NH HI AS NM MD XS --outSAMunmapped Within --outFilterMultimapNmax 50
Expression estimation with RSEM rsem-calculate-expression v1.2.31 --alignments --no-bam-output
Differential expression estimation with edgeR v3.28.0
Assembly: GRCm39
Supplementary files format and content: .csv

source_name: KrasG12D;Tp53R172H; Pdx1-Cre Cells (KPC)

growth_protocol: Cells were grown in DMEM with 10% FBS at 37°C and 5.3% CO2

extract_protocol:
Total RNA was extracted using the manufacturers recommendations for the Rneasy protocol
RNA libraries were prepared for sequencing using standard Truseq Illumina protocols

value: Pancreas ductal adenocarcinoma

value: AKO KPC


title: WGBS_TSC1_TET3KO

data_processing:
Raw reads were subjected to adapter and quality trimming using cutadapt (version 2.4; parameters: --quality-cutoff 20 --overlap 5 --minimum-length 25; Illumina TruSeq adapter clipped from both reads), followed by trimming of 10 and 5 nucleotides from the 5’ and 3’ end of the first read and 15 and 5 nucleotides from the 5’ and 3’ end of the second read.
The trimmed reads were aligned to the mouse genome (mm10) using BSMAP (version 2.90; parameters: -v 0.1 -s 16 -q 20 -w 100 -S 1 -u -R).
Duplicates were removed using the ‘MarkDuplicates’ command from GATK (version 4.1.4.1; --VALIDATION_STRINGENCY=LENIENT --REMOVE_DUPLICATES=true).
Methylation rates were called using mcall from the MOABS package (version 1.3.2; default parameters).
All analyses were restricted to autosomes and only CpGs covered by at least 10 and at most 150 reads were considered for downstream analyses.
Assembly: mm10
Supplementary files format and content: Bigwig file containing methylation rates for CpGs covered by at least 10 and at most 150 reads: <chr> <start> <end> <methylation rate>

growth_protocol: TSC Tet3 KO clone G11 (background TSC1 line) was cultured in TSC medium (RPMI+GlutaMAX (Thermo, #61870044), 20 % fetal bovine serum (PAN, #P30-2602), 1 mM sodium pyruvate (Thermo, #11360070), 100 µM 2-mercaptoethanol (Thermo, #21985023) and 1X Penicillin/Streptomycin (Thermo, #15140122); 25 ng/ml FGF4 (R&D systems, #235-F4-025) and 1 µg/ml Heparin (Sigma, #H3149).

extract_protocol:
The DNA was extracted using the PureLink Genomic DNA Mini Kit (Thermo, #K182002) according to manufacturer’s instructions.
Concentration of genomic DNA (gDNA) was quantified using a Qubit 3.0 Fluorometer. The DNA was sheared in Covaris micro TUBE AFA Fiber Pre-Slit Snap-Cap tubes (SKU: 520045) and cleaned up with the Zymo DNA Clean & Concentrator-5 kit (#D4013) following manufacturer’s guidelines. Sheared gDNA was bisulfite converted following manufacturer's guidelines with the EZ DNA Methylation-Gold Kit (Zymo #D5005), and libraries were prepared using the Accel-NGS Methyl-seq DNA library kit (Swift Biosciences, #30024-SWI). Libraries were cleaned using Agencourt AMPure XP beads (Beckman Coulter, #A63881), and the absence of adapters was confirmed on the Agilent TapeStation HS D5000. The final libraries were sequenced on a NovaSeq platform (Illumina) yielding 150 bp paired-end reads.

treatment_protocol: TSCs wildtype cells (TSC1 line) were transfected with Cas9/sgRNA system targeting the Tet3 gene.

tag: condition

value: TET3 KO


title: cDNA_pLenti6_pool1_IP_rep2

description:
NIH3T3 cells transfected with a pool of 12 cDNA pLenti6 plasmids.
cDNAExpV2IPpool1_AlignedReads.bam.txDT.rds
TableS1.xlsx


title: AKO_3

value: KrasG12D;Tp53R172H; Pdx1-Cre Cells (KPC)


title: ChIPseq_ESC_WT_EED_Input_Rep2


title: Calm3_distToPolyA_100_input_rep1

description:
MCF7 cells transfected with Calm3 contruct with 100nt distance between the DRACH motif and polyA site, input, rep1
TableS1.xlsx

value: human cell line, plasmid with mouse sequence

value: human breast cancer

value: Plasmid transfection with an insert described in the 'description'. Insert sequences are in TableS1.xlsx.


title: cDNA_pLenti6_pool3_input_rep2

description:
NIH3T3 cells transfected with a pool of 12 cDNA pLenti6 plasmids.
cDNAExpV2INPpool3_AlignedReads.bam.txDT.rds
TableS1.xlsx


title: AKO_1


title: AKO_5


title: cDNA_pLenti6_pool3_input_rep1


title: cDNA_pLenti6_pool1_IP_rep1


title: Calm3_distToPolyA_150_input_rep1

description:
MCF7 cells transfected with Calm3 contruct with 150nt distance between the DRACH motif and polyA site, input, rep1
TableS1.xlsx


title: cDNA_pLenti6_pool2_IP_rep1


title: ChIPseq_TSC1_WT_EED_Input_Rep1


title: WT_6

value: WT KPC


title: ChIPseq_ESC_WT_EED_Rep2


title: cDNA_pLenti6_pool2_input_rep2

description:
NIH3T3 cells transfected with a pool of 12 cDNA pLenti6 plasmids.
cDNAExpV2INPpool2_AlignedReads.bam.txDT.rds
TableS1.xlsx


title: WT_5


title: cDNA_pLenti6_pool2_input_rep1


title: WT_3


title: WT_4


title: Calm3_distToPolyA_150_input_rep2

description:
MCF7 cells transfected with Calm3 contruct with 150nt distance between the DRACH motif and polyA site, input, rep2
TableS1.xlsx


title: cDNA_pLenti6_pool1_input_rep1

description:
NIH3T3 cells transfected with a pool of 12 cDNA pLenti6 plasmids.
cDNAExpV2INPpool1_AlignedReads.bam.txDT.rds
TableS1.xlsx


title: cDNA_pLenti6_pool3_IP_rep1


title: Mettl3Inhibitor_actD_9h_rep2

description:
HEK293T cells treated with Mettl3 inhibitor or untreated, and treated with actD for 0,3,6,9h.
actD_Mettl3inh.bed

treatment_protocol: For combined treatment with Actinomycin D and m6A inhibitor STM2457 (DC Chemicals, Shanghai, #DC53045), the cells were seeded into 60-mm dishes and grown for one day until 50% confluent. Then, STM2457 (Yankova et al. 2021) compound was added at a final concentration of 5µM. After 6 hours of incubation, Actinomycin D was added (7.5µM) for the indicated time points and the cells were collected after a total treatment of 15 hours with STM2457.

value: human

value: human embryonic kidney

value: Mettl3 inhibitor STM2457 treatment (15h) and actD timepoints 9h, rep2


title: AKO_4


title: WT_1


title: WT_2


title: Mettl3Inhibitor_actD_6h_rep2

value: Mettl3 inhibitor STM2457 treatment (15h) and actD timepoints 6h, rep2


title: Mettl3Inhibitor_actD_3h_rep2

value: Mettl3 inhibitor STM2457 treatment (15h) and actD timepoints 3h, rep3


title: dRS rep1

first: Hasan

last: Khatib

address: 1675 Observatory Dr.

department: Animal and Dairy Sciences

institute: University of Wisconsin-Madison

data_processing:
Basecalls performed using bcl2fastq v2.20.0.422
Sequenced reads were trimmed for low-quality and adaptor sequences using Trimmomatic v.0.39 , then mapped to mm39 whole genome using STAR v.2.7.4a  with parameters –outSAMmapqUnique 60 --outFilterType BySJout --outSAMstrandField intronMotif --quantMode GeneCounts --twopassMode Basic
For gene-level expression, gene counts were estimated using “--quantMode GeneCounts” option in STAR (Dobin et al., 2013).
Assembly: GRCm39
Supplementary files format and content: Matrix table with raw gene counts for every gene and every sample

source_name: Left ventricular myocardium

extract_protocol:
Total RNA was extracted from mouse left ventricles at the age of 2 month using TRIzol Reagent (Life technologies, Thermo Fisher Scientific, Waltham, MA) according to the manufacturer’s protocol. DNase I (#D9905K, Lucigen, Middleton, WI) treatment was applied to clean up genomic DNA before RNA-seq. The quantity and quality were determined by Nanodrop One (Thermo Fisher Scientific, Waltham, MA) and electrophoresis.
RNA libraries (Illumina TruSeq standard Total RNA Gold) were prepared for sequencing using standard Illumina protocols.

value: heart, left ventricular myocardium

value: dRS

value: 2 month


title: dRS rep2


title: AKO_2


title: dRS rep3


title: Mettl3Inhibitor_actD_9h_rep1

value: Mettl3 inhibitor STM2457 treatment (15h) and actD timepoints 9h, rep1


title: Wildtype rep3

value: WT


title: Mettl3Inhibitor_actD_6h_rep1

value: Mettl3 inhibitor STM2457 treatment (15h) and actD timepoints 6h, rep1


title: Wildtype rep2


title: cDNA_pLenti6_pool1_input_rep2

value: mouse, human

value: mouse fibroblast

value: Plasmid transfection with cDNA pLentli6 plasmid pool described in Table S1.


title: Mettl3Inhibitor_actD_0h_rep2

value: Mettl3 inhibitor STM2457 treatment (15h) and actD timepoints 0h, rep2


title: Mettl3Inhibitor_actD_0h_rep1

value: Mettl3 inhibitor STM2457 treatment (15h) and actD timepoints 0h, rep1


title: Wildtype rep1


title: RNAseq_TSC1_WT_EZH2i_7d_acute_response_Rep2

description: RNA sequencing of TSCs

data_processing:
Raw reads were subjected to adapter and quality trimming with cutadapt (version 2.4; parameters: --quality-cutoff 20 --overlap 5 --minimum-length 25 --interleaved --adapter AGATCGGAAGAGC -A AGATCGGAAGAGC), followed by poly-A trimming with cutadapt (parameters: --interleaved --overlap 20 --minimum-length --adapter "A[100]" --adapter "T[100]").
Reads were aligned to the mouse reference genome (mm10) using STAR (version 2.7.5a; parameters: --runMode alignReads --chimSegmentMin 20 --outSAMstrandField intronMotif --quantMode GeneCounts) and transcripts were quantified using stringtie (version 2.0.6; parameters: -e) with GENCODE annotation (release VM19).
Assembly: mm10
Supplementary files format and content: Tab-delimited file containing gene abundances: <Gene ID> <Gene Name> <Reference> <Strand> <Start> <End> <Coverage> <FPKM> <TPM>

growth_protocol: Trophoblast stem cells (TSCs) cells were grown on MEF-coated cell culture dishes containing TSC medium (RPMI+GlutaMAX (Thermo, #61870044), 20 % fetal bovine serum (PAN, #P30-2602), 1 mM sodium pyruvate (Thermo, #11360070), 100 µM 2-mercaptoethanol (Thermo, #21985023) and 1X Penicillin/Streptomycin (Thermo, #15140122); 25 ng/ml FGF4 (R&D systems, #235-F4-025) and 1 µg/ml Heparin (Sigma, #H3149). Splitting was carried out every five to seven days by rinsing the cells once with DPBS (Thermo, #14190144) before detaching the cells using Trypsin-EDTA (0.05%)(Thermo, #25300054). TSCs were passaged in clumps. Before sample collection, TSCs were passaged at least one passage without MEFs to dilute out feeder cells. During this time, the cells were cultured in MEF-conditioned TSC medium (70 % MEF-conditioned medium, 30 % TSC medium, +FGF4 (37.5 ng/ml), Heparin (1.5 µg/ml)).

extract_protocol:
Cell pellets were resuspended in 350 µl RLT Plus buffer containing 1% 2-mercaptoethanol (Thermo, #21985023). After cell lysis by trituration and vortexing, RNA was extracted using RNeasy Plus Micro Kit (Qiagen, #74034) and RNA concentration and quality was measured using the Agilent RNA Screen Tape (Agilent Technologies, #5067- 5576) on an Agilent 4150 Tapestation system. All samples analyzed had a RINe value higher than 8,0, and were subsequently used for library preparation.
mRNA libraries were prepared using KAPA Stranded RNA-Seq Kit (KapaBiosystem, #KK8421/07962207001) according to the manufacturer’s instructions. 500 ng of total RNA was used for each sample to enter the library preparation protocol. For adapter ligation dual indexes were used (NEXTFLEX® Unique Dual Index Barcodes #NOVA-514150 and #NOVA-514151) at a working concentration of 71 nM (5 µl of 1 µM stock in each 70 µl ligation reaction). Twelve library PCR cycles were used. Quality and concentration of the obtained libraries were measured using Agilent High Sensitivity D5000 ScreenTape (Agilent-Technologies, #5067- 5592) on an Agilent 4150 TapeStation.

treatment_protocol: TSCs were cultured on MEFs in TSC medium containing either (1) Dnmt1 inhibitor (GSK-3484862; 25 dissolved in DMSO to 1 mM) at a final concentration of 1 μM, (2) EZH2 inhibitor (Tazemetostat/EPZ6438, Biovision, #2383-5, dissolved in DMSO to 10 mM) at a final concentration of 10 µM, (3) Both inhibitors (DNMT1i and EZH2i, same concentrations as in (1) and (2)) or (4) DMSO, equal volume as in the "Both inhibitor" condition for up to seven days. The medium was changed every day and inhibitors/DMSO freshly added to the medium prior to media change. Before sample collection, TSCs were passaged at least once without MEFs in MEF-conditioned medium (+inhibitor/DMSO) to dilute out feeder cells. For the collection after two days of DNMT1i, cells were MEF depleted just before starting the treatment to avoid MEF contamination at the time of collection. After 7 days of DNMT1 inhibition, a recovery period of 14 days started by splitting the cells and maintenance of TSCs in standard conditions (in the absence of DNMT1i/DMSO) for 14 days.

value: Trophoblast stem cells


title: Mettl3Inhibitor_actD_3h_rep1

value: human

value: Mettl3 inhibitor STM2457 treatment (15h) and actD timepoints 3h, rep1


title: RNAseq_TSC1_WT_DNMT1i_EZH2i_7d_acute_response_Rep2

description: RNA sequencing of TSCs

value: DNMT1i + EZH2i


title: RNAseq_TSC1_WT_DNMT1i_EZH2i_7d_acute_response_Rep1

description: RNA sequencing of TSCs


title: spliceLongMPRE_IP_rep2

description:
RNA amplicon
HEK293T cells transfected with MPRE, m6Aseq input sample, rep4
BC_counts_raw_spliceTwist.csv
TableS3.csv

data_processing:
Fastq files were processed with a custom R script. Barcodes matching the planned barcode were counted for each sample. See Table S3 for the planned barcode sequences.
Assembly: MPRE sequences based on mm9 and Assembly: hg19, no alignment was performed
Supplementary files format and content: Csv file BC_counts_raw_spliceTwist.csv containing raw barcode counts of each construct in the MPRE for input and IP of both biological relicates. Excel file (TableS3.xlsx) containing all planned construct sequences and description.

extract_protocol:
RNA was extracted with NucleoZOL (Macherey-Nagel)
RNA was poly-A selected with oligo dT-beads (Dynabeads® mRNA DIRECT™ Kit life tech). After mild fragmentation, samples were divided into input and IP sample, and IP samples were subjected to m6A immunoprecipitation. RNA from both the input and IP samples was reverse transcribed with a sequence specific RT primer to include the 10 nt unique barcode sequence of each construct in the MPRE. PCR amplicons of these fragments were generated.

treatment_protocol: HEK293T cells were seeded in each well of a 60 mm plate. 24 hours later cells were transfected with Polyjet reagent. Cells were harvested 24 hours post transfection.

value: human, mouse, synthetic

value: human embryonic kidney

value: MPRE plasmids transfection, MPRE contains ~2000 mouse, human and synthetic sequences


title: spliceLongMPRE_IP_rep1

description:
RNA amplicon
HEK293T cells transfected with MPRE, m6Aseq input sample, rep3
BC_counts_raw_spliceTwist.csv
TableS3.csv


title: spliceLongMPRE_input_rep1

description:
RNA amplicon
HEK293T cells transfected with MPRE, m6Aseq input sample, rep1
BC_counts_raw_spliceTwist.csv
TableS3.csv


title: WT_rep3_IP_additional

description:
MCF7 cells transfected with Calm3, Nol12 and Coa3 DRACH WT, rep3, IP
TableS1.xlsx

extract_protocol:
RNA was extracted with BIO TRI RNA reagent (Bio-lab)
RNA was poly-A selected with oligo dT-beads (Dynabeads® mRNA DIRECT™ Kit life tech) and subjected to m6Aseq2 according to Dierks et al, 2021 Nature Methods.

value: human cell line, plasmid with mouse sequence

value: human breast cancer

value: Plasmid transfection with an insert described in the 'description'. Insert sequences are in TableS1.xlsx.


title: RNAseq_TSC1_WT_DNMT1i_7d_recovery_14d_Rep1

description: RNA sequencing of TSCs

value: WT


title: RNAseq_TSC1_WT_DNMT1i_7d_acute_response_Rep2

description: RNA sequencing of TSCs


title: RNAseq_TSC1_WT_EZH2i_7d_acute_response_Rep1

description: RNA sequencing of TSCs


title: spliceLongMPRE_input_rep2

description:
RNA amplicon
HEK293T cells transfected with MPRE, m6Aseq input sample, rep2
BC_counts_raw_spliceTwist.csv
TableS3.csv


title: WT_rep1_IP_additional

description:
MCF7 cells transfected with Calm3, Nol12 and Coa3 DRACH WT, rep1, IP
TableS1.xlsx


title: RNAseq_TSC1_WT_DNMT1i_7d_acute_response_Rep1

description: RNA sequencing of TSCs


title: nointron_rep3_IP_additional

description:
MCF7 cells transfected with Calm3, Nol12 and Coa3 DRACH no intron, rep3, IP
TableS1.xlsx


title: RNAseq_TSC1_WT_DNMT1i_2d_Rep1

description: RNA sequencing of TSCs


title: RNAseq_TSC1_WT_DNMT1i_7d_Rep2

description: RNA sequencing of TSCs


title: nointron_rep2_IP_additional

description:
MCF7 cells transfected with Calm3, Nol12 and Coa3 DRACH no intron, rep2, IP
TableS1.xlsx


title: mutant_rep3_IP_additional

description:
MCF7 cells transfected with Calm3, Nol12 and Coa3 DRACH mutants, rep3, IP
TableS1.xlsx


title: RNAseq_TSC1_WT_DMSO_7d_acute_response_Rep2

description: RNA sequencing of TSCs

value: DMSO


title: RNAseq_TSC1_WT_DMSO_7d_Rep2

description: RNA sequencing of TSCs


title: WT_rep2_input_additional

description:
MCF7 cells transfected with Calm3, Nol12 and Coa3 DRACH WT, rep2, input
TableS1.xlsx


title: nointron_rep1_IP_additional

description:
MCF7 cells transfected with Calm3, Nol12 and Coa3 DRACH mutants, rep1, IP
TableS1.xlsx


title: RNAseq_TSC1_WT_DNMT1i_7d_Rep1

description: RNA sequencing of TSCs


title: RNAseq_TSC1_WT_DNMT1i_2d_Rep2

description: RNA sequencing of TSCs


title: mutant_rep2_IP_additional

description:
MCF7 cells transfected with Calm3, Nol12 and Coa3 DRACH mutants, rep2, IP
TableS1.xlsx


title: RNAseq_TSC1_WT_DNMT1i_7d_recovery_14d_Rep2

description: RNA sequencing of TSCs


title: WT_rep3_input_additional

description:
MCF7 cells transfected with Calm3, Nol12 and Coa3 DRACH WT, rep3, input
TableS1.xlsx


title: RNAseq_TSC1_WT_DMSO_7d_acute_response_Rep1

description: RNA sequencing of TSCs


title: RNAseq_TSC1_WT_DMSO_7d_Rep1

description: RNA sequencing of TSCs


title: nointron_rep1_input_additional

description:
MCF7 cells transfected with Calm3, Nol12 and Coa3 DRACH mutants, rep1, input
TableS1.xlsx


title: HiC_ESC_WT_Rep2

data_processing:
Raw reads were subjected to adapter and quality trimming with cutadapt (version 2.4; parameters: --quality-cutoff 20 --overlap 5 --minimum-length 25 --adapter AGATCGGAAGAGC -A AGATCGGAAGAGC).
Mates were separately aligned to the mouse genome (mm10) using bwa with the ‘mem’ command (version 0.7.17; parameters: -A 1 -B 4 -E 50 -L 0).
Hi-C matrices for each replicate were built using HiCExplorer with the ‘hicBuildMatrix’ command (version 3.6; parameters: --binSize 5000 --restrictionSequence GATC --danglingSequence GATC --minMappingQuality 30).
TSC and ESC replicates were merged respectively using ‘hicSumMatrices’. TSC and ESC matrices were normalized together using ‘hicNormalize’ (parameters: --smallest) and corrected using ‘hicCorrectMatrix’ (parameters: --correctionMethod KR).
For whole-chromosome representation and compartment analysis bins of matrices at 5 kb resolution were merged into 100 kb bins using ‘hicMergeMatrixBins’ (parameters: -nb 20).
Assembly: mm10
Supplementary files format and content: Normalized and KR-corrected matrices of 5 kb resolution in h5 format.

growth_protocol: Embryonic stem cells (ESC) cells were grown on gelatin/MEF-coated cell culture dishes containing ESC medium (Knockout DMEM (Thermo, #10829018, 15% fetal bovine serum (PAN, #P30-2602), 1X GlutaMAX supplement (Thermo, #35050-038), 1X non-essential amino acids (Thermo, #11140-035), 100 µM 2-mercaptoethanol (Thermo, #21985023), 1X Penicillin/Streptomycin (Thermo, #15140122) and 1X leukemia inhibitory factor (LIF) (selfmade). Splitting was carried out every three days by rinsing the cells once with DPBS (Thermo, #14190144) before detaching the cells using TryplE (Gibco, #12604013). Before sample collection, ESCs were passaged at least one passage without MEFs to dilute out feeder cells. During this time, the cells were cultured in ESC medium containing 2X LIF. Trophoblast stem cells (TSCs) cells were grown on MEF-coated cell culture dishes containing TSC medium (RPMI+GlutaMAX (Thermo, #61870044), 20 % fetal bovine serum (PAN, #P30-2602), 1 mM sodium pyruvate (Thermo, #11360070), 100 µM 2-mercaptoethanol (Thermo, #21985023) and 1X Penicillin/Streptomycin (Thermo, #15140122); 25 ng/ml FGF4 (R&D systems, #235-F4-025) and 1 µg/ml Heparin (Sigma, #H3149). Splitting was carried out every five to seven days by rinsing the cells once with DPBS (Thermo, #14190144) before detaching the cells using Trypsin-EDTA (0.05%)(Thermo, #25300054). TSCs were passaged in clumps. Before sample collection, TSCs were passaged at least one passage without MEFs to dilute out feeder cells. During this time, the cells were cultured in MEF-conditioned TSC medium (70 % MEF-conditioned medium, 30 % TSC medium, +FGF4 (37.5 ng/ml), Heparin (1.5 µg/ml)).

extract_protocol:
To extract the nuclei, cells were incubated on ice for 10 min with ice-cold Lysis Buffer (50mM Tris HCl pH 7.5, 150 mM NaCl, 5 mM 788 EDTA, 0.5% NP-40, 1.15% Triton X-100, 25X Protease Inhibitor in Milli-Q Water). Extracted nuclei were centrifuged at 750g for 5 min at 4°C, two times washed with 1X DPBS, snap frozen and stored at -80°C.
Nuclei were first permeabilized using 0.5% SDS at 62°C for 10 min and later, chromatin was digested with DpnII (NEB, #R0543) for 90 min at 37°C with gentle rotation. The overhangs generated by digestion were filled and marked with biotin-14-dATP (Thermo, #19524016) by a 90 min incubation at 37°C with gentle rotation. After, DNA fragments were ligated for 4 hours at 20°C with gentle rotation using T4 DNA Ligase (Thermo, #M0202M). Chromatin was then reverse-crosslinked, precipitated and sheared with Covaris S220 (2 cycles, each 50sec long; 10% duty; 4 intensity; 200 cycles/burst). The biotin-marked-DNA shared fragments were pulled down using Dynabeads MyOne Streptavidin T1 beads (Thermo, #65601), purified and further processed for Illumina sequencing with NEBNext Ultra II Library Prep Kit for Illumina according to the kit guidelines (NEBNext End Prep, Adaptor Ligation, PCR enrichment of Adaptor-Ligated DNA using NEBNext Multiplex Oligos for Illumina). Clean up and size selection were performed with AMPure beads.

treatment_protocol: 2x10^6 cells were dissociated in a single-cell-suspension using pre-warmed Trypsin-EDTA (0.05%) and incubated for 5-10 min at 37°C. Trypsin was blocked by adding 10% FCS/DPBS and cells were centrifuged for 5 min at 1100 rpm. Cell pellet was resuspended in a 2% PFA (in 10%FCS/DPBS) fixation solution and incubated at room temperature for 10 min while tumbling. The reaction was quenced on ice by adding Glycine (final concentration 125mM) and cells were collected by centrifugation at 400 g for 8 min at 4°C.

value: Embryonic stem cells


title: HiC_TSC1_WT_Rep1


title: nointron_rep3_input_additional

description:
MCF7 cells transfected with Calm3, Nol12 and Coa3 DRACH no intron, rep3, input
TableS1.xlsx


title: mutant_rep3_input_additional

description:
MCF7 cells transfected with Calm3, Nol12 and Coa3 DRACH mutants, rep3, input
TableS1.xlsx


title: HiC_TSC1_WT_Rep2


title: nointron_rep2_input_additional

description:
MCF7 cells transfected with Calm3, Nol12 and Coa3 DRACH no intron, rep2, input
TableS1.xlsx


title: HiC_ESC_WT_Rep1


title: mutant_rep1_input_additional


title: mutant_rep1_IP_additional


title: mESC_SLC25A3_WT_input

description: mESC cells transfected with SLC25A WT, input

data_processing:
Reads were mapped to mm9 (mESC) genome assembly and the transfected GFP-SLC25A3 plasmid with STAR/2.7.9a (Dobin et al., 2013). Per-base read coverages were obtained with txtools (https://github.com/AngelCampos/txtools)
mm9, transfected GFP-SLC25A3 plasmid (See TableS1 for sequence)
R data table (rds) file output from txtools https://github.com/AngelCampos/txtools. In addition, the TableS1.xlsx excel file contains the tranfected plasmid sequences.

source_name: mESC

growth_protocol: V6.5 mESC line was kept in DMEM (Gibco) supplemented with 1% Penicillin-Streptomycin, 1 mM L-glutamine, 1% non-essential amino acids, 20% high-grade fetal bovine serum (Biological Industries), beta-mercaptoethanol and 10 µg recombinant leukemia inhibiting factor. Cells were kept on tissue culture plates covered by gelatin (0.2%), in co-culture with in-house generated, radiation-inactivated mouse embryonic fibroblasts.

treatment_protocol: Cells were seeded on gelatin-covered 6-well plates (without MEF feeder cells) for transfection. GFP-SLC25A3 plasmid was transfected with the TransIT-X2 reagent (Mirus) according to the manufacturer’s instructions. Cells were collected 24 hours post-transfection.

value: mouse cell line, plasmid with human sequence

value: mESC

value: V6.5 mouse embryonic stem cell


title: WT_rep2_IP_additional

description:
MCF7 cells transfected with Calm3, Nol12 and Coa3 DRACH WT, rep2, IP
TableS1.xlsx


title: mESC_SLC25A3_mutMain_IP

description: mESC cells transfected with SLC25A mut-main, m6A IP


title: WT_rep1_input_additional

description:
MCF7 cells transfected with Calm3, Nol12 and Coa3 DRACH WT, rep1, input
TableS1.xlsx


title: MCF7_MR25_input_rep2

description: MCF7 cells transfected with MR25, m6A input sample, replicate 2, part of experiment 2

data_processing:
Reads were mapped to hg19 genome assembly and the transfected plasmid sequences with STAR/2.7.9a (Dobin et al., 2013). Per-base read coverages were obtained with txtools (https://github.com/AngelCampos/txtools)
hg19 and the transfected plasmid (See TableS1 for sequence)
R data table (rds) file output from txtools https://github.com/AngelCampos/txtools. In addition, the TableS1.xlsx excel file contains the tranfected plasmid sequences.


title: MCF_MR25_IP_rep2

description: MCF7 cells transfected with MR25, m6A IP sample, replicate 2, part of experiment 2


title: MCF7_MR25_input_rep1

description: MCF7 cells transfected with MR25, m6A input sample, replicate 1, part of experiment 2


title: mESC_SLC25A3_mutMain_input

description: mESC cells transfected with SLC25A mut-main, input


title: MCF7_MR29MR30MR31_E1_input_rep2

description: MCF7 cells transfected with MR29, MR30 and MR31, input sample, replicate 2, part of experiment 1


title: mESC_SLC25A3_mutAll_IP

description: mESC cells transfected with SLC25A mut-all, m6A IP


title: mESC_SLC25A3_mutSecondary_input

description: mESC cells transfected with SLC25A mut-secondary, input


title: MCF_MR25_IP_rep1

description: MCF7 cells transfected with MR25, m6A IP sample, replicate 1, part of experiment 2


title: mESC_SLC25A3_mutSecondary_IP

description: mESC cells transfected with SLC25A mut-secondary, m6A IP


title: MCF7_MR29MR30MR31_E1_IP_rep2

description: MCF7 cells transfected with MR29, MR30 and MR31, m6A IP sample, replicate 2, part of experiment 1


title: RNAseq_TSC3_EEDKO_Rep2

description: RNA sequencing of TSCs

data_processing:
Raw reads were subjected to adapter and quality trimming with cutadapt (version 2.4; parameters: --quality-cutoff 20 --overlap 5 --minimum-length 25 --interleaved --adapter AGATCGGAAGAGC -A AGATCGGAAGAGC), followed by poly-A trimming with cutadapt (parameters: --interleaved --overlap 20 --minimum-length --adapter "A[100]" --adapter "T[100]").
Reads were aligned to the mouse reference genome (mm10) using STAR (version 2.7.5a; parameters: --runMode alignReads --chimSegmentMin 20 --outSAMstrandField intronMotif --quantMode GeneCounts) and transcripts were quantified using stringtie (version 2.0.6; parameters: -e) with GENCODE annotation (release VM19).
Genome_build: mm10
Supplementary_files_format_and_content: Tab-delimited file containing gene abundances: <Gene ID> <Gene Name> <Reference> <Strand> <Start> <End> <Coverage> <FPKM> <TPM>

treatment_protocol: On the day of collection, TSCs were rinsed once with DPBS (Thermo, #14190144) and detached using Trypsin-EDTA (0.05%)(Thermo, #25300054). The cells were spun down for 4 min at 1000 rpm, counted and flash frozen on dry ice. The cell pellets were stored at -80°C.

value: EED KO


title: mESC_SLC25A3_mutAll_input

description: mESC cells transfected with SLC25A mut-all, input


title: MCF7_MR17_E1_input_rep2

description: MCF7 cells transfected with MR17, input sample, replicate 2, part of experiment 1


title: mutant_rep2_input_additional

description:
MCF7 cells transfected with Calm3, Nol12 and Coa3 DRACH mutants, rep2, input
TableS1.xlsx


title: RNAseq_TSC3_EEDKO_Rep1

description: RNA sequencing of TSCs

value: Trophoblast stem cells


title: mESC_SLC25A3_WT_IP

description: mESC cells transfected with SLC25A WT, m6A IP


title: MCF7_MR17_E1_IP_rep2

description: MCF7 cells transfected with MR17, m6A IP sample, replicate 2, part of experiment 1


title: MCF7_MR15_E1_IP_rep2

description: MCF7 cells transfected with MR15, m6A IP sample, replicate 2, part of experiment 1


title: MCF7_MR3MR6MR9_E1_input_rep2

description: MCF7 cells transfected with MR3, MR6 and MR9, input sample, replicate 2, part of experiment 1


title: RNAseq_TSC2_3BKO_Rep1

description: RNA sequencing of TSCs

value: DNMT3B KO


title: RNAseq_TSC2_3BKO_Rep2

description: RNA sequencing of TSCs


title: MCF7_MR15_E1_input_rep2

description: MCF7 cells transfected with MR15, input sample, replicate 2, part of experiment 1


title: RNAseq_TSC1_WT_Rep1

description: RNA sequencing of TSCs


title: RNAseq_TSC1_WT_Rep2

description: RNA sequencing of TSCs


title: MCF7_MR29MR30MR31_E1_input_rep1

description: MCF7 cells transfected with MR29, MR30 and MR31, input sample, replicate 1, part of experiment 1


title: MCF7_MR17_E1_input_rep1

description: MCF7 cells transfected with MR17, input sample, replicate 1, part of experiment 1


title: RNAseq_TSC1_WT_EZH2i_5w_recovery_4w_Rep2

description: RNA sequencing of TSCs

growth_protocol: Trophoblast stem cells (TSCs) cells were grown on MEF-coated cell culture dishes containing TSC medium (RPMI+GlutaMAX (Thermo, #61870044), 20 % fetal bovine serum (PAN, #P30-2602), 1 mM sodium pyruvate (Thermo, #11360070), 100 µM 2-mercaptoethanol (Thermo, #21985023) and 1X Penicillin/Streptomycin (Thermo, #15140122); 25 ng/ml FGF4 (R&D systems, #235-F4-025) and 1 µg/ml Heparin (Sigma, #H3149)). The EZH2 inhibitor (Tazemetostat/EPZ6438, Biovision, #2383-5, dissolved in DMSO to 10 mM) was freshly added to the TSC medium at a final concentration of 10 µM. The medium was changed every day. The cells were grown five weeks in the presence of the EZH2i. Subsequently, the inhibitor was removed by splitting the cells, and the cells were cultivated for four weeks with standard conditions for recovery. Splitting was carried out every five to seven days by rinsing the cells once with DPBS (Thermo, #14190144) before detaching the cells using Trypsin-EDTA (0.05%)(Thermo, #25300054). TSCs were passaged in clumps. Before sample collection, TSCs were passaged at least one passage without MEFs to dilute out feeder cells. During this time, the cells were cultured in MEF-conditioned TSC medium (70 % MEF-conditioned medium, 30 % TSC medium, +FGF4 (37.5 ng/ml), Heparin (1.5 µg/ml)).


title: RNAseq_TSC1_WT_EZH2i_5w_recovery_1w_Rep1

description: RNA sequencing of TSCs

growth_protocol: Trophoblast stem cells (TSCs) cells were grown on MEF-coated cell culture dishes containing TSC medium (RPMI+GlutaMAX (Thermo, #61870044), 20 % fetal bovine serum (PAN, #P30-2602), 1 mM sodium pyruvate (Thermo, #11360070), 100 µM 2-mercaptoethanol (Thermo, #21985023) and 1X Penicillin/Streptomycin (Thermo, #15140122); 25 ng/ml FGF4 (R&D systems, #235-F4-025) and 1 µg/ml Heparin (Sigma, #H3149)). The EZH2 inhibitor (Tazemetostat/EPZ6438, Biovision, #2383-5, dissolved in DMSO to 10 mM) was freshly added to the TSC medium at a final concentration of 10 µM. The medium was changed every day. The cells were grown five weeks in the presence of the EZH2i. Subsequently, the inhibitor was removed by splitting the cells, and the cells were cultivated for one week with standard conditions for recovery. Splitting was carried out every five to seven days by rinsing the cells once with DPBS (Thermo, #14190144) before detaching the cells using Trypsin-EDTA (0.05%)(Thermo, #25300054). TSCs were passaged in clumps. Before sample collection, TSCs were passaged at least one passage without MEFs to dilute out feeder cells. During this time, the cells were cultured in MEF-conditioned TSC medium (70 % MEF-conditioned medium, 30 % TSC medium, +FGF4 (37.5 ng/ml), Heparin (1.5 µg/ml)).


title: MCF7_MR29MR30MR31_E1_IP_rep1

description: MCF7 cells transfected with MR29, MR30 and MR31, m6A IP sample, replicate 1, part of experiment 1


title: MCF7_MR15_E1_input_rep1

description: MCF7 cells transfected with MR15, input sample, replicate 1, part of experiment 1


title: RNAseq_TSC1_WT_EZH2i_5w_recovery_4w_Rep1

description: RNA sequencing of TSCs


title: RNAseq_TSC1_WT_EZH2i_5w_Rep2

description: RNA sequencing of TSCs

growth_protocol: Trophoblast stem cells (TSCs) cells were grown on MEF-coated cell culture dishes containing TSC medium (RPMI+GlutaMAX (Thermo, #61870044), 20 % fetal bovine serum (PAN, #P30-2602), 1 mM sodium pyruvate (Thermo, #11360070), 100 µM 2-mercaptoethanol (Thermo, #21985023) and 1X Penicillin/Streptomycin (Thermo, #15140122); 25 ng/ml FGF4 (R&D systems, #235-F4-025) and 1 µg/ml Heparin (Sigma, #H3149)). The EZH2 inhibitor (Tazemetostat/EPZ6438, Biovision, #2383-5, dissolved in DMSO to 10 mM) was freshly added to the TSC medium at a final concentration of 10 µM. The medium was changed every day. The cells were grown five weeks in the presence of the EZH2i. Splitting was carried out every five to seven days by rinsing the cells once with DPBS (Thermo, #14190144) before detaching the cells using Trypsin-EDTA (0.05%)(Thermo, #25300054). TSCs were passaged in clumps. Before sample collection, TSCs were passaged at least one passage without MEFs to dilute out feeder cells. During this time, the cells were cultured in MEF-conditioned TSC medium (70 % MEF-conditioned medium, 30 % TSC medium, +FGF4 (37.5 ng/ml), Heparin (1.5 µg/ml)).


title: MCF7_MR3MR6MR9_E1_input_rep1

description: MCF7 cells transfected with MR3, MR6 and MR9, input sample, replicate 1, part of experiment 1


title: RNAseq_TSC1_WT_EZH2i_5w_recovery_1w_Rep2

description: RNA sequencing of TSCs


title: RNAseq_TSC1_WT_EZH2i_5w_Rep1

description: RNA sequencing of TSCs


title: MCF7_MR3MR6MR9_E1_IP_rep1

description: MCF7 cells transfected with MR3, MR6 and MR9, m6A IP sample, replicate 1, part of experiment 1


title: MCF7_MR15_E1_IP_rep1

description: MCF7 cells transfected with MR15, m6A IP sample, replicate 1, part of experiment 1


title: RNAseq_TSC1_WT_DMSO_5w_Rep1

description: RNA sequencing of TSCs

growth_protocol: Trophoblast stem cells (TSCs) cells were grown on MEF-coated cell culture dishes containing TSC medium (RPMI+GlutaMAX (Thermo, #61870044), 20 % fetal bovine serum (PAN, #P30-2602), 1 mM sodium pyruvate (Thermo, #11360070), 100 µM 2-mercaptoethanol (Thermo, #21985023) and 1X Penicillin/Streptomycin (Thermo, #15140122); 25 ng/ml FGF4 (R&D systems, #235-F4-025) and 1 µg/ml Heparin (Sigma, #H3149)). DMSO (equal volume as EZH2i, 1:1000) was freshly added to the TSC medium. The medium was changed every day. The cells were grown five weeks in the presence of the DMSO. Splitting was carried out every five to seven days by rinsing the cells once with DPBS (Thermo, #14190144) before detaching the cells using Trypsin-EDTA (0.05%)(Thermo, #25300054). TSCs were passaged in clumps. Before sample collection, TSCs were passaged at least one passage without MEFs to dilute out feeder cells. During this time, the cells were cultured in MEF-conditioned TSC medium (70 % MEF-conditioned medium, 30 % TSC medium, +FGF4 (37.5 ng/ml), Heparin (1.5 µg/ml)).


title: RNAseq_TSC1_WT_DMSO_5w_Rep2

description: RNA sequencing of TSCs


title: MCF7_MR17_E1_IP_rep1

description: MCF7 cells transfected with MR17, m6A IP sample, replicate 1, part of experiment 1


title: MCF7_MR1MR4MR7_IP_rep2

description: MCF7 cells transfected with MR1, MR4 and MR7, m6A IP sample, replicate 2, part of experiment 2


title: MCF7_MR1MR4MR7_input_rep2

description: MCF7 cells transfected with MR1, MR4 and MR7, m6A input sample, replicate 2, part of experiment 2


title: RNAseq_TSC1_RNF2KO_Rep1

description: RNA sequencing of TSCs

value: RNF2 KO


title: RNAseq_TSC1_KDM2BKO_Rep2

description: RNA sequencing of TSCs

value: KDM2B KO


title: RNAseq_TSC1_KDM2BKO_Rep1

description: RNA sequencing of TSCs


title: MCF7_MR84_IP_rep2

description: MCF7 cells transfected with MR84, m6A IP sample, replicate 2, part of experiment 2


title: MCF7_MR1MR4MR7_IP_rep1

description: MCF7 cells transfected with MR1, MR4 and MR7, m6A IP sample, replicate 1, part of experiment 2


title: MINUTE_TSC3_EEDKO_Input_Pool2_Rep2

description: Quantitative ChIP of TSCs

data_processing:
Read pairs matching parts of the adaptor sequence (SBS3 or T7 promoter) in either read1 or read2 were removed using cutadapt v3.2.
Reads were demultiplexed using cutadapt v3.2 allowing only one mismatch per barcode and written into sample-specific FASTQ files used for subsequent mapping.
Sample-specific paired FASTQ files were mapped to the reference mm10 using bowtie2 v2.3.5.1 with --fast and --reorder parameter. Alignments were processed into sorted BAM files and replicates were pooled using samtools v1.10.
Duplicate reads are marked using UMI-sensitive deduplication tool je-suite (v2.0.RC) (https://github.com/gbcs-embl/Je/). Read pairs are marked as duplicates if their read1 (first-in-pair) sequences have the same UMI (allowing for 1 mismatch) and map to the same location in the genome. Blacklisted regions as downloaded from ENCODE were then removed from BAM files using bedtools v2.30.
Input coverage tracks with 1 bp resolution in bigwig format were generated from BAM files using deepTools v3.5.0 bamCoverage and scaled to a reads-per-genome-coverage of one (1xRPGC, also referred to as ‘1x normalization’) using the mm10 effective genome size. ChIP coverage tracks were generated from BAM files using deepTools (v3.5.0) bamCoverage. Quantitative scaling of the ChIP-Seq tracks amongst conditions within each pool was based on their Input-Normalized Mapped Read Count (INRC). INRC was calculated by dividing the number of unique reference-mapped reads by the respective number of Input reads: #mapped[ChIP] / #mapped[Input]. This essentially corrects for an uneven representation of barcodes in the Input. It has been previously shown that INRCs are proportional to the amount of epitope present in each condition (8). Reference condition (TSC WT) was scaled to 1x coverage (also termed Reads per Genome Coverage, RPGC). All other conditions within the same pool were scaled relative to the reference using the ratio of INRCs multiplied by the scaling factor determined for 1x normalization of the reference: ( #mapped[ChIP] / #mapped[Input] ) / ( #mapped[ChIP_Reference] / #mapped[Input_Reference] ) * scaling factor.
Genome_build: mm10
Supplementary_files_format_and_content: Bigwig files are scaled according to MINUTE quantification with respect to the reference of each pool (TSC1 WT, 1xRPGC)

extract_protocol:
Native cell pellets were lysed and digested with MNase to enrich for mononucleosome population. The digestion was quenched by EGTA-containing end-repair and ligation buffer, in which each sample was ligated to adaptor molecules carrying unique barcode. Ligation was quenched by EDTA-containing lysis dilution buffer, before combining all samples in one tube. After centrifugation, pool supernatant was recovered and aliquoted for individual ChIP. 2 x 10^6 cell-equivalent of pool supernatant was used for ChIP against each histone modification, with the anti-H3K4me3 (Millipore #04-745), anti-H3K27me3 (Cell Signaling #9733) and anti-H2AK119ub (Cell Signaling #8240) antibodies precoupled to Protein A magnetic beads.
After thorough washes, ChIP DNA was recovered with Proteinase K treatment and purified for linear amplification by in vitro transcription. The RNA product was then ligated to a pre-adenylated RNA 3’ adaptor (RA3), which served as a primer binding site for reverse transcription. The resulting cDNA was purified and used as a template for library PCR with barcoded primers compatible with Illumina sequencing platform. Typically, 1-2 x 10^5 cell equivalent of pool supernatant was used as Input, which is subjected to the same experimental workflow for library construction as the ChIP DNA. All nucleic-acid purification were carried out with AMPure SPRI size selection method (Beckman Coulter). Library size distribution was assessed by Agilent BioAnalyzer and were quantified by Qubit DNA high sensitivity assay before dilution for sequencing on a NovaSeq 6000 platform.

treatment_protocol: On the day of collection, TSCs were rinsed once with DPBS (Thermo, #14190144) and detached using Trypsin-EDTA (0.05%)(Thermo, #25300054). The cells were spun down for 4 min at 1000 rpm, counted and flash frozen on dry ice.The cell pellets were stored at -80°C.


title: MINUTE_TSC3_EEDKO_Input_Pool2_Rep3

description: Quantitative ChIP of TSCs


title: MCF7_MR84_input_rep2

description: MCF7 cells transfected with MR84, m6A input sample, replicate 2, part of experiment 2


title: MINUTE_TSC3_EEDKO_Input_Pool2_Rep1

description: Quantitative ChIP of TSCs


title: MCF7_MR3MR6MR9_E1_IP_rep2

description: MCF7 cells transfected with MR3, MR6 and MR9, m6A IP sample, replicate 2, part of experiment 1


title: MCF7_MR84_IP_rep1

description: MCF7 cells transfected with MR84, m6A IP sample, replicate 1, part of experiment 2


title: MCF7_MR84_input_rep1

description: MCF7 cells transfected with MR84, m6A input sample, replicate 1, part of experiment 2


title: MINUTE_TSC3_EEDKO_H3K4me3_Pool2_Rep2

description: Quantitative ChIP of TSCs


title: RNAseq_TSC1_RNF2KO_Rep2

description: RNA sequencing of TSCs


title: MCF7_MR2MR5MR8_IP_rep2

description: MCF7 cells transfected with MR2, MR5 and MR8, m6A IP sample, replicate 2, part of experiment 2


title: MINUTE_TSC3_EEDKO_H3K4me3_Pool2_Rep1

description: Quantitative ChIP of TSCs


title: MCF7_MR2MR5MR8_input_rep2

description: MCF7 cells transfected with MR2, MR5 and MR8, m6A input sample, replicate 2, part of experiment 2


title: MINUTE_TSC3_EEDKO_H3K4me3_Pool2_Rep3

description: Quantitative ChIP of TSCs


title: MINUTE_TSC3_EEDKO_H3K27me3_Pool2_Rep2

description: Quantitative ChIP of TSCs


title: MCF7_MR2MR5MR8_IP_rep1

description: MCF7 cells transfected with MR2, MR5 and MR8, m6A IP sample, replicate 1, part of experiment 2


title: MCF7_MR2MR5MR8_input_rep1

description: MCF7 cells transfected with MR2, MR5 and MR8, m6A input sample, replicate 1, part of experiment 2


title: MINUTE_TSC3_EEDKO_H2AK119ub1_Pool2_Rep2

description: Quantitative ChIP of TSCs


title: MCF7_MR3MR6MR9_input_rep2

description: MCF7 cells transfected with MR3, MR6 and MR9, m6A input sample, replicate 2, part of experiment 2


title: MINUTE_TSC3_EEDKO_H3K27me3_Pool2_Rep1

description: Quantitative ChIP of TSCs


title: MINUTE_TSC3_EEDKO_H2AK119ub1_Pool2_Rep3

description: Quantitative ChIP of TSCs


title: MCF7_MR3MR6MR9_IP_rep1

description: MCF7 cells transfected with MR3, MR6 and MR9, m6A IP sample, replicate 1, part of experiment 2


title: MCF7_MR3MR6MR9_input_rep1

description: MCF7 cells transfected with MR3, MR6 and MR9, m6A input sample, replicate 1, part of experiment 2


title: MINUTE_TSC3_EEDKO_H2AK119ub1_Pool2_Rep1

description: Quantitative ChIP of TSCs


title: MCF7_MR1MR4MR7_input_rep1

description: MCF7 cells transfected with MR1, MR4 and MR7, m6A input sample, replicate 1, part of experiment 2


title: MCF7_MR3MR6MR9_IP_rep2

description: MCF7 cells transfected with MR3, MR6 and MR9, m6A IP sample, replicate 2, part of experiment 2


title: MINUTE_TSC2_3BKO_Input_Pool1_Rep2

description: Quantitative ChIP of TSCs


title: MINUTE_TSC2_3BKO_Input_Pool1_Rep3

description: Quantitative ChIP of TSCs


title: MCF7_MR83_input_rep2

description: MCF7 cells transfected with MR83, m6A input sample, replicate 2, part of experiment 2


title: MCF7_MR83_IP_rep2

description: MCF7 cells transfected with MR83, m6A IP sample, replicate 2, part of experiment 2


title: MCF7_MR83_IP_rep1

description: MCF7 cells transfected with MR83, m6A IP sample, replicate 1, part of experiment 2


title: MINUTE_TSC2_3BKO_Input_Pool1_Rep1

description: Quantitative ChIP of TSCs


title: MINUTE_TSC2_3BKO_H3K4me3_Pool1_Rep3

description: Quantitative ChIP of TSCs


title: MCF7_MR83_input_rep1

description: MCF7 cells transfected with MR83, m6A input sample, replicate 1, part of experiment 2


title: MINUTE_TSC3_EEDKO_H3K27me3_Pool2_Rep3

description: Quantitative ChIP of TSCs


title: MINUTE_TSC2_3BKO_H3K4me3_Pool1_Rep2

description: Quantitative ChIP of TSCs


title: MCF7_MR38_IP_rep2

description: MCF7 cells transfected with MR38, m6A IP sample, replicate 2, part of experiment 2


title: MINUTE_TSC2_3BKO_H3K4me3_Pool1_Rep1

description: Quantitative ChIP of TSCs


title: MCF7_MR38_IP_rep1

description: MCF7 cells transfected with MR38, m6A IP sample, replicate 1, part of experiment 2


title: MINUTE_TSC2_3BKO_H3K27me3_Pool1_Rep2

description: Quantitative ChIP of TSCs


title: MINUTE_TSC2_3BKO_H3K27me3_Pool1_Rep3

description: Quantitative ChIP of TSCs


title: MCF7_MR38_input_rep1

description: MCF7 cells transfected with MR38, m6A input sample, replicate 1, part of experiment 2


title: MINUTE_TSC2_3BKO_H3K27me3_Pool1_Rep1

description: Quantitative ChIP of TSCs


title: MCF7_MR36_input_rep2

description: MCF7 cells transfected with MR36, m6A input sample, replicate 2, part of experiment 2


title: MCF7_MR36_IP_rep1

description: MCF7 cells transfected with MR36, m6A IP sample, replicate 1, part of experiment 2


title: MINUTE_TSC2_3BKO_H2AK119ub1_Pool1_Rep3

description: Quantitative ChIP of TSCs


title: MINUTE_TSC2_3BKO_H2AK119ub1_Pool1_Rep1

description: Quantitative ChIP of TSCs


title: MCF7_MR36_input_rep1

description: MCF7 cells transfected with MR36, m6A input sample, replicate 1, part of experiment 2


title: MINUTE_TSC1_WT_Input_Pool2_Rep2

description: Quantitative ChIP of TSCs


title: MCF7_MR35_input_rep2

description: MCF7 cells transfected with MR35, m6A input sample, replicate 2, part of experiment 2


title: MINUTE_TSC1_WT_Input_Pool2_Rep1

description: Quantitative ChIP of TSCs


title: MINUTE_TSC2_3BKO_H2AK119ub1_Pool1_Rep2

description: Quantitative ChIP of TSCs


title: MCF7_MR35_input_rep1

description: MCF7 cells transfected with MR35, m6A input sample, replicate 1, part of experiment 2


title: MCF7_MR35_IP_rep1

description: MCF7 cells transfected with MR35, m6A IP sample, replicate 1, part of experiment 2


title: MCF7_MR35_IP_rep2

description: MCF7 cells transfected with MR35, m6A IP sample, replicate 2, part of experiment 2


title: MCF7_MR40_IP_rep2

description: MCF7 cells transfected with MR40, m6A IP sample, replicate 2, part of experiment 2


title: MINUTE_TSC1_WT_Input_Pool1_Rep3

description: Quantitative ChIP of TSCs


title: MCF7_MR40_input_rep2

description: MCF7 cells transfected with MR40, m6A input sample, replicate 2, part of experiment 2


title: MINUTE_TSC1_WT_Input_Pool1_Rep1

description: Quantitative ChIP of TSCs


title: MINUTE_TSC1_WT_H3K4me3_Pool2_Rep3

description: Quantitative ChIP of TSCs


title: MCF7_MR40_IP_rep1

description: MCF7 cells transfected with MR40, m6A IP sample, replicate 1, part of experiment 2


title: MINUTE_TSC1_WT_Input_Pool1_Rep2

description: Quantitative ChIP of TSCs


title: MCF7_MR38_input_rep2

description: MCF7 cells transfected with MR38, m6A input sample, replicate 2, part of experiment 2


title: MCF7_MR36_IP_rep2

description: MCF7 cells transfected with MR36, m6A IP sample, replicate 2, part of experiment 2


title: MINUTE_TSC1_WT_H3K4me3_Pool2_Rep2

description: Quantitative ChIP of TSCs


title: MCF7_MR32MR33MR34_IP_rep2

description: MCF7 cells transfected with MR32, MR33 and MR34, m6A IP sample, replicate 2, part of experiment 2


title: MINUTE_TSC1_WT_H3K4me3_Pool2_Rep1

description: Quantitative ChIP of TSCs


title: MCF7_MR32MR33MR34_input_rep2

description: MCF7 cells transfected with MR32, MR33 and MR34, m6A input sample, replicate 2, part of experiment 2


title: MINUTE_TSC1_WT_H3K4me3_Pool1_Rep2

description: Quantitative ChIP of TSCs


title: MCF7_MR32MR33MR34_IP_rep1

description: MCF7 cells transfected with MR32, MR33 and MR34, m6A IP sample, replicate 1, part of experiment 2


title: MINUTE_TSC1_WT_Input_Pool2_Rep3

description: Quantitative ChIP of TSCs


title: MINUTE_TSC1_WT_H3K4me3_Pool1_Rep1

description: Quantitative ChIP of TSCs


title: MCF7_MR32MR33MR34_input_rep1

description: MCF7 cells transfected with MR32, MR33 and MR34, m6A input sample, replicate 1, part of experiment 2


title: MCF7_MR76_92_input_rep2

description: MCF7 cells transfected with MR76 with intron and additional 92 bases insert, m6A input sample, replicate 2, part of experiment 3


title: MCF7_MR76NI92_input_rep2

description: MCF7 cells transfected with MR76 no intron and additional 92 bases insert, m6A input sample, replicate 2, part of experiment 3


title: MINUTE_TSC1_WT_H3K27me3_Pool2_Rep2

description: Quantitative ChIP of TSCs


title: MINUTE_TSC1_WT_H3K27me3_Pool1_Rep3

description: Quantitative ChIP of TSCs


title: MINUTE_TSC1_WT_H3K27me3_Pool2_Rep3

description: Quantitative ChIP of TSCs


title: MCF7_MR76_140_input_rep2

description: MCF7 cells transfected with MR76 with intron and additional 140 bases insert, m6A input sample, replicate 2, part of experiment 3


title: MINUTE_TSC1_WT_H3K27me3_Pool2_Rep1

description: Quantitative ChIP of TSCs


title: MINUTE_TSC1_WT_H3K27me3_Pool1_Rep2

description: Quantitative ChIP of TSCs


title: MCF7_MR76NI_input2_rep2

description: MCF7 cells transfected with MR76 no intron, m6A input sample technical replicate 2, biological replicate 2, part of experiment 3


title: MCF7_MR76NI140_input_rep2

description: MCF7 cells transfected with MR76 no intron and additional 140 bases insert, m6A input sample, replicate 2, part of experiment 3


title: MINUTE_TSC1_WT_H3K27me3_Pool1_Rep1

description: Quantitative ChIP of TSCs


title: MINUTE_TSC1_WT_H2AK119ub1_Pool2_Rep2

description: Quantitative ChIP of TSCs


title: MINUTE_TSC1_WT_H2AK119ub1_Pool2_Rep3

description: Quantitative ChIP of TSCs


title: MINUTE_TSC1_WT_H3K4me3_Pool1_Rep3

description: Quantitative ChIP of TSCs


title: MCF7_MR40_input_rep1

description: MCF7 cells transfected with MR40, m6A input sample, replicate 1, part of experiment 2


title: MCF7_MR76_input1_rep2

description: MCF7 cells transfected with MR76 with intron, m6A input sample technical replicate 1, biological replicate 2, part of experiment 3


title: MCF7_MR76NI_input1_rep2

description: MCF7 cells transfected with MR76 no intron, m6A input sample technical replicate 1, biological replicate 2, part of experiment 3


title: MINUTE_TSC1_WT_H2AK119ub1_Pool2_Rep1

description: Quantitative ChIP of TSCs


title: MCF7_MR74_input_rep2

description: MCF7 cells transfected with MR74 with intron, m6A input sample, replicate 2, part of experiment 3


title: MINUTE_TSC1_WT_H2AK119ub1_Pool1_Rep2

description: Quantitative ChIP of TSCs


title: MCF7_MR74NI_input_rep2

description: MCF7 cells transfected with MR74 no intron, m6A input sample, replicate 2, part of experiment 3


title: MINUTE_TSC1_WT_H2AK119ub1_Pool1_Rep3

description: Quantitative ChIP of TSCs


title: MINUTE_TSC1_WT_H2AK119ub1_Pool1_Rep1

description: Quantitative ChIP of TSCs


title: MCF7_MR73_input_rep2

description: MCF7 cells transfected with MR73 with intron, m6A input sample, replicate 2, part of experiment 3


title: MCF7_MR72_input_rep2

description: MCF7 cells transfected with MR72 with intron, m6A input sample, replicate 2, part of experiment 3


title: MCF7_MR73NI_input_rep2

description: MCF7 cells transfected with MR73 no intron, m6A input sample, replicate 2, part of experiment 3


title: MCF7_MR72NI_input_rep2

description: MCF7 cells transfected with MR72 no intron, m6A input sample, replicate 2, part of experiment 3


title: MINUTE_TSC1_WT_EZH2i_5w_recovery_4w_Input_Pool2_Rep1

description: Quantitative ChIP of TSCs


title: MCF7_MR76_140_input_rep1

description: MCF7 cells transfected with MR76 with intron and additional 140 bases insert, m6A input sample, replicate 1, part of experiment 3


title: MCF7_MR76_input2_rep2

description: MCF7 cells transfected with MR76 with intron, m6A input sample technical replicate 2, biological replicate 2, part of experiment 3


title: MINUTE_TSC1_WT_EZH2i_5w_recovery_4w_H3K4me3_Pool2_Rep3

description: Quantitative ChIP of TSCs


title: MCF7_MR76NI92_input_rep1

description: MCF7 cells transfected with MR76 no intron and additional 92 bases insert, m6A input sample, replicate 1, part of experiment 3


title: MINUTE_TSC1_WT_EZH2i_5w_recovery_4w_H3K4me3_Pool2_Rep1

description: Quantitative ChIP of TSCs


title: MCF7_MR76_92_input_rep1

description: MCF7 cells transfected with MR76 with intron and additional 92 bases insert, m6A input sample, replicate 1, part of experiment 3


title: MINUTE_TSC1_WT_EZH2i_5w_recovery_4w_H3K27me3_Pool2_Rep3

description: Quantitative ChIP of TSCs


title: MINUTE_TSC1_WT_EZH2i_5w_recovery_4w_H3K27me3_Pool2_Rep1

description: Quantitative ChIP of TSCs


title: MCF7_MR76_input2_rep1

description: MCF7 cells transfected with MR76 with intron, m6A input sample technical replicate 2, biological replicate 1, part of experiment 3


title: MINUTE_TSC1_WT_EZH2i_5w_recovery_4w_H3K27me3_Pool2_Rep2

description: Quantitative ChIP of TSCs


title: MINUTE_TSC1_WT_EZH2i_5w_recovery_4w_H2AK119ub1_Pool2_Rep3

description: Quantitative ChIP of TSCs


title: MINUTE_TSC1_WT_EZH2i_5w_recovery_4w_H2AK119ub1_Pool2_Rep2

description: Quantitative ChIP of TSCs


title: MINUTE_TSC1_WT_EZH2i_5w_recovery_4w_H2AK119ub1_Pool2_Rep1

description: Quantitative ChIP of TSCs


title: MINUTE_TSC1_WT_EZH2i_5w_recovery_4w_Input_Pool2_Rep3

description: Quantitative ChIP of TSCs


title: MINUTE_TSC1_RNF2KO_Input_Pool2_Rep3

description: Quantitative ChIP of TSCs


title: MCF7_MR73_input_rep1

description: MCF7 cells transfected with MR73 with intron, m6A input sample, replicate 1, part of experiment 3


title: MCF7_MR73NI_input_rep1

description: MCF7 cells transfected with MR73 no intron, m6A input sample, replicate 1, part of experiment 3


title: MCF7_MR76_input1_rep1

description: MCF7 cells transfected with MR76 with intron, m6A input sample technical replicate 1, biological replicate 1, part of experiment 3


title: MINUTE_TSC1_RNF2KO_Input_Pool2_Rep2

description: Quantitative ChIP of TSCs


title: MCF7_MR74_input_rep1

description: MCF7 cells transfected with MR74 with intron, m6A input sample, replicate 1, part of experiment 3


title: MINUTE_TSC1_RNF2KO_Input_Pool2_Rep1

description: Quantitative ChIP of TSCs


title: MCF7_MR72_input_rep1

description: MCF7 cells transfected with MR72 with intron, m6A input sample, replicate 1, part of experiment 3


title: MINUTE_TSC1_WT_EZH2i_5w_recovery_4w_Input_Pool2_Rep2

description: Quantitative ChIP of TSCs


title: MINUTE_TSC1_WT_EZH2i_5w_recovery_4w_H3K4me3_Pool2_Rep2

description: Quantitative ChIP of TSCs


title: MINUTE_TSC1_RNF2KO_H3K4me3_Pool2_Rep3

description: Quantitative ChIP of TSCs


title: MINUTE_TSC1_RNF2KO_H3K4me3_Pool2_Rep1

description: Quantitative ChIP of TSCs


title: MCF7_MR76NI140_input_rep1

description: MCF7 cells transfected with MR76 no intron and additional 140 bases insert, m6A input sample, replicate 1, part of experiment 3


title: MCF7_MR72NI_input_rep1

description: MCF7 cells transfected with MR72 no intron, m6A input sample, replicate 1, part of experiment 3


title: MCF7_MR76_92_IP_rep2

description: MCF7 cells transfected with MR76 with intron and additional 92 bases insert, m6A IP sample, replicate 2, part of experiment 3


title: MCF7_MR76_140_IP_rep2

description: MCF7 cells transfected with MR76 with intron and additional 140 bases insert, m6A IP sample, replicate 2, part of experiment 3


title: MCF7_MR76NI_input2_rep1

description: MCF7 cells transfected with MR76 no intron, m6A input sample technical replicate 2, biological replicate 1, part of experiment 3


title: MCF7_MR76NI_input1_rep1

description: MCF7 cells transfected with MR76 no intron, m6A input sample technical replicate 1, biological replicate 1, part of experiment 3


title: MINUTE_TSC1_RNF2KO_H3K27me3_Pool2_Rep3

description: Quantitative ChIP of TSCs


title: MCF7_MR76NI92_IP_rep2

description: MCF7 cells transfected with MR76 no intron and additional 92 bases insert, m6A IP sample, replicate 2, part of experiment 3


title: MINUTE_TSC1_RNF2KO_H3K27me3_Pool2_Rep2

description: Quantitative ChIP of TSCs


title: MCF7_MR74NI_input_rep1

description: MCF7 cells transfected with MR74 no intron, m6A input sample, replicate 1, part of experiment 3


title: MCF7_MR76NI140_IP_rep2

description: MCF7 cells transfected with MR76 no intron and additional 140 bases insert, m6A IP sample, replicate 2, part of experiment 3


title: MINUTE_TSC1_RNF2KO_H3K27me3_Pool2_Rep1

description: Quantitative ChIP of TSCs


title: MINUTE_TSC1_RNF2KO_H2AK119ub1_Pool2_Rep3

description: Quantitative ChIP of TSCs


title: MCF7_MR76NI_IP1_rep2

description: MCF7 cells transfected with MR76 no intron, m6A IP sample technical replicate 1, biological replicate 2, part of experiment 3


title: MINUTE_TSC1_RNF2KO_H2AK119ub1_Pool2_Rep2

description: Quantitative ChIP of TSCs


title: MCF7_MR76_IP2_rep2

description: MCF7 cells transfected with MR76 with intron, m6A IP sample technical replicate 2, biological replicate 2, part of experiment 3


title: MINUTE_TSC1_RNF2KO_H2AK119ub1_Pool2_Rep1

description: Quantitative ChIP of TSCs


title: MCF7_MR74_IP_rep2

description: MCF7 cells transfected with MR74 with intron, m6A IP sample, replicate 2, part of experiment 3


title: MCF7_MR76_IP1_rep2

description: MCF7 cells transfected with MR76 with intron, m6A IP sample technical replicate 1, biological replicate 2, part of experiment 3


title: MINUTE_TSC1_KDM2BKO_Input_Pool2_Rep2

description: Quantitative ChIP of TSCs


title: MINUTE_TSC1_KDM2BKO_Input_Pool2_Rep3

description: Quantitative ChIP of TSCs


title: MINUTE_TSC1_KDM2BKO_Input_Pool2_Rep1

description: Quantitative ChIP of TSCs


title: MCF7_MR73_IP_rep2

description: MCF7 cells transfected with MR73 with intron, m6A IP sample, replicate 2, part of experiment 3


title: MINUTE_TSC1_KDM2BKO_H3K4me3_Pool2_Rep3

description: Quantitative ChIP of TSCs


title: MINUTE_TSC1_RNF2KO_H3K4me3_Pool2_Rep2

description: Quantitative ChIP of TSCs


title: MCF7_MR73NI_IP_rep2

description: MCF7 cells transfected with MR73 no intron, m6A IP sample, replicate 2, part of experiment 3


title: MCF7_MR74NI_IP_rep2

description: MCF7 cells transfected with MR74 no intron, m6A IP sample, replicate 2, part of experiment 3


title: MINUTE_TSC1_KDM2BKO_H3K4me3_Pool2_Rep2

description: Quantitative ChIP of TSCs


title: MINUTE_TSC1_KDM2BKO_H3K4me3_Pool2_Rep1

description: Quantitative ChIP of TSCs


title: MCF7_MR72_IP_rep2

description: MCF7 cells transfected with MR72 with intron, m6A IP sample, replicate 2, part of experiment 3


title: MINUTE_TSC1_KDM2BKO_H3K27me3_Pool2_Rep2

description: Quantitative ChIP of TSCs


title: MINUTE_TSC1_KDM2BKO_H3K27me3_Pool2_Rep3

description: Quantitative ChIP of TSCs


title: MCF7_MR76_140_IP_rep1

description: MCF7 cells transfected with MR76 with intron and additional 140 bases insert, m6A IP sample, replicate 1, part of experiment 3


title: MINUTE_TSC1_KDM2BKO_H3K27me3_Pool2_Rep1

description: Quantitative ChIP of TSCs


title: MCF7_MR76NI_IP2_rep2

description: MCF7 cells transfected with MR76 no intron, m6A IP sample technical replicate 2, biological replicate 2, part of experiment 3


title: MCF7_MR76NI92_IP_rep1

description: MCF7 cells transfected with MR76 no intron and additional 92 bases insert, m6A IP sample, replicate 1, part of experiment 3


title: MINUTE_TSC1_KDM2BKO_H2AK119ub1_Pool2_Rep2

description: Quantitative ChIP of TSCs


title: MINUTE_TSC1_KDM2BKO_H2AK119ub1_Pool2_Rep3

description: Quantitative ChIP of TSCs


title: MCF7_MR76NI_IP2_rep1

description: MCF7 cells transfected with MR76 no intron, m6A IP sample technical replicate 2, biological replicate 1, part of experiment 3


title: MCF7_MR76NI140_IP_rep1

description: MCF7 cells transfected with MR76 no intron and additional 140 bases insert, m6A IP sample, replicate 1, part of experiment 3


title: MCF7_MR76_IP2_rep1

description: MCF7 cells transfected with MR76 with intron, m6A IP sample technical replicate 2, biological replicate 1, part of experiment 3


title: MINUTE_TSC1_KDM2BKO_H2AK119ub1_Pool2_Rep1

description: Quantitative ChIP of TSCs


title: MCF7_MR76NI_IP1_rep1

description: MCF7 cells transfected with MR76 no intron, m6A IP sample technical replicate 1, biological replicate 1, part of experiment 3


title: MINUTE_ESC_WT_Input_Pool1_Rep1

description: Quantitative ChIP of ESCs

growth_protocol: Embryonic stem cells (ESC) cells were grown on gelatin/MEF-coated cell culture dishes containing ESC medium (Knockout DMEM (Thermo, #10829018, 15% fetal bovine serum (PAN, #P30-2602), 1X GlutaMAX supplement (Thermo, #35050-038), 1X non-essential amino acids (Thermo, #11140-035), 100 µM 2-mercaptoethanol (Thermo, #21985023), 1X Penicillin/Streptomycin (Thermo, #15140122) and 1X leukemia inhibitory factor (LIF) (selfmade). Splitting was carried out every three days by rinsing the cells once with DPBS (Thermo, #14190144) before detaching the cells using TryplE (Gibco, #12604013). Before sample collection, ESCs were passaged at least one passage without MEFs to dilute out feeder cells. During this time, the cells were cultured in ESC medium containing 2X LIF.

treatment_protocol: On the day of collection, ESCs were rinsed once with DPBS (Thermo, #14190144) and detached using TryplE (Gibco, #12604013). The cells were spun down for 4 min at 1000 rpm, counted and flash frozen on dry ice. The cell pellets were stored at -80°C.


title: MINUTE_ESC_WT_Input_Pool1_Rep3

description: Quantitative ChIP of ESCs


title: MINUTE_ESC_WT_Input_Pool1_Rep2

description: Quantitative ChIP of ESCs


title: MCF7_MR76_IP1_rep1

description: MCF7 cells transfected with MR76 with intron, m6A IP sample technical replicate 1, biological replicate 1, part of experiment 3


title: MINUTE_ESC_WT_H3K4me3_Pool1_Rep3

description: Quantitative ChIP of ESCs


title: MINUTE_ESC_WT_H3K4me3_Pool1_Rep2

description: Quantitative ChIP of ESCs


title: MCF7_MR74NI_IP_rep1

description: MCF7 cells transfected with MR74 no intron, m6A IP sample, replicate 1, part of experiment 3


title: MCF7_MR76_92_IP_rep1

description: MCF7 cells transfected with MR76 with intron and additional 92 bases insert, m6A IP sample, replicate 1, part of experiment 3


title: MCF7_MR73_IP_rep1

description: MCF7 cells transfected with MR73 with intron, m6A IP sample, replicate 1, part of experiment 3


title: MCF7_MR74_IP_rep1

description: MCF7 cells transfected with MR74 with intron, m6A IP sample, replicate 1, part of experiment 3


title: MCF7_MR72NI_IP_rep2

description: MCF7 cells transfected with MR72 no intron, m6A IP sample, replicate 2, part of experiment 3